Sample records for 2c receptor agonists

The serotonin 5-HT2Creceptor has shown promise in vivo as a pharmacotherapeutic target for alcoholism. For example, recently, a novel 4-phenyl-2-N,N-dimethylaminotetralin (PAT) drug candidate, that demonstrates 5-HT2Creceptoragonist activity together with 5-HT2A/2B receptor inverse agonist activity, was shown to reduce operant responding for ethanol after peripheral administration to rats. Previous studies have shown that the 5-HT2Creceptor is found throughout the mesoaccumbens pathway and that 5-HT2Creceptor agonism causes activation of ventral tegmental area (VTA) GABA neurons. It is unknown what effect 5-HT2Creceptor modulation has on GABA release in the nucleus accumbens core (NAcc). To this end, microdialysis coupled to capillary electrophoresis with laser-induced fluorescence was used to quantify extracellular neurotransmitter concentrations in the NAcc under basal and after potassium stimulation conditions, in response to PAT analogs and other 5-HT2Creceptor modulators administered by reverse dialysis to rats. 5-HT2Creceptoragonists specifically attenuated stimulated GABA release in the NAcc while 5-HT2C antagonists or inverse agonists had no effect. Agents with activity at 5-HT2A receptors had no effect on GABA release. Thus, in contrast to results reported for the VTA, current results suggest 5-HT2Creceptoragonists decrease stimulated GABA release in the NAcc, and provide a possible mechanism of action for 5HT2C-mediated negative modulation of ethanol self-administration. PMID:25382408

The serotonin 2C (5-HT2C) receptor has been identified as a potential drug target for the treatment of a variety of central nervous system (CNS) disorders, such as obesity, substance abuse, and schizophrenia. In this Viewpoint article, recent progress in developing selective 5-HT2Cagonists for use in treating these disorders is summarized, including the work of our group. Challenges in this field and the possible future directions are described. Homology modeling as a method to predict the binding modes of 5-HT2C ligands to the receptor is also discussed. Compared to known ligands, the improved pharmacological profiles of the 2-phenylcyclopropylmethylamine-based 5-HT2Cagonists make them preferred candidates for further studies. PMID:26507582

The neurotransmitter 5-hydroxytryptamine (5-HT; serotonin) has long been associated with the control of a variety of motivated behaviors, including feeding. Much of the evidence linking 5-HT and feeding behavior was obtained from studies of the effects of the 5-HT releaser (dex)fenfluramine in laboratory animals and humans. Recently, the selective 5-HT2Creceptoragonist lorcaserin received FDA approval for the treatment of obesity. This review examines evidence to support the use of selective 5-HT2Creceptoragonists as treatments for conditions beyond obesity, including substance abuse (particularly nicotine, psychostimulant, and alcohol dependence), obsessive compulsive, and excessive gambling disorder. Following a brief survey of the early literature supporting a role for 5-HT in modulating food and drug reinforcement, we propose that intrinsic differences between SSRI and serotonin releasers may have underestimated the value of serotonin-based pharmacotherapeutics to treat clinical forms of addictive behavior beyond obesity. We then highlight the critical involvement of the 5-HT2Creceptor in mediating the effect of (dex)fenfluramine on feeding and body weight gain and the evidence that 5-HT2Creceptoragonists reduce measures of drug reward and impulsivity. A recent report of lorcaserin efficacy in a smoking cessation trial further strengthens the idea that 5-HT2Creceptoragonists may have potential as a treatment for addiction. This review was prepared as a contribution to the proceedings of the 11th International Society for Serotonin Research Meeting held in Hermanus, South Africa, July 9-12, 2014. PMID:25870913

Heterocycle-fused azepines are discussed as potent 5-HT2Creceptoragonists with excellent selectivity over 5-HT2B agonism. Synthesis and structure activity relationships are outlined for a series of bicyclic pyridazino[3,4-d]azepines. By comparison with earlier published work, in vitro assays predict a high probability for achieving CNS penetration for a potent and selective compound 15a, a pre-requisite to achieve in vivo efficacy. PMID:27381086

A series of pyrido[3,4-d]azepines that are potent and selective 5-HT2Creceptoragonists is disclosed. Compound 7 (PF-04781340) is identified as a suitable lead owing to good 5-HT2C potency, selectivity over 5-HT2B agonism, and in vitro ADME properties commensurate with an orally available and CNS penetrant profile. The synthesis of a novel bicyclic tetrasubstituted pyridine core template is outlined, including rationale to account for the unexpected formation of aminopyridine 13 resulting from an ammonia cascade cyclization. PMID:25815155

Obesity continues to be a burgeoning health problem worldwide. Before their removal from the market, fenfluramine and the more active enantiomer dexfenfluramine were considered to be among the most effective of weight loss agents. Much of the weight loss produced by fenfluramine was attributed to the direct activation of serotonin 5-HT(2C) receptors in the central nervous system via the desmethyl-metabolite of fenfluramine, norfenfluramine. Norfenfluramine, however, is non-selective, activating additional serotonin receptors, such as 5-HT(2A) and 5-HT(2B), which likely mediated the heart valve hypertrophy seen in many patients. Development of highly selective 5-HT(2C) agonists may recapitulate the clinical anti-obesity properties observed with fenfluramine while avoiding the significant cardiovascular and pulmonary side effects. PMID:16249524

Extensive behavioral and biochemical evidence suggests an agonist role at the 5-HT2A receptor, and perhaps the 5-HT2Creceptor, in the mechanism of action of hallucinogenic drugs. However the published in vitro pharmacological properties of N,N-dimethyltryptamine (DMT), an hallucinogenic tryptamine analog, are not consistent with this hypothesis. We, therefore, undertook an extensive investigation into the properties of DMT at 5-HT2A and 5-HT2Creceptors. In fibroblasts transfected with the 5-HT2A receptor or the 5-HT2Creceptor, DMT activated the major intracellular signaling pathway (phosphoinositide hydrolysis) to an extent comparable to that produced by serotonin. Because drug efficacy changes with receptor density and cellular microenvironment, we also examined the properties of DMT in native preparations using a behavioral and biochemical approach. Rats were trained to discriminate an antagonist ketanserin from an agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) in a two-lever choice paradigm. Pharmacological studies showed that responding on the DOI and ketanserin lever reflected agonist and antagonist activity at 5-HT2A receptors, and hence, was a suitable model for evaluating the in vivo functional properties of DMT. Like other 5-HT2A receptoragonists, DMT substituted fully for DOI. Intact choroid plexus was used to evaluate the agonist properties at endogenous 5-HT2Creceptors; DMT was a partial agonist at 5-HT2Creceptors in this native preparation. Thus, we conclude that DMT behaves as an agonist at both 5-HT2A and 5-HT2A receptors. One difference was evident in that the 5-HT2C, but not the 5-HT2A, receptor showed a profound desensitization to DMT over time. This difference is interesting in light of the recent report that the hallucinogenic activity of DMT does not tolerate in humans and suggests the 5-HT2Creceptor plays a less prominent role in the action of DMT. PMID:9768567

There is some controversy as to whether 5-HT(2C) receptoragonists are anxiogenic or anxiolytic. The effects of the novel 5-HT(2C) receptoragonist, (S)-2-chloro-5-fluoro-indol-1-yl)-1-methyl ethylamine fumarate (RO 60 0175), in three models of anxiety were therefore tested. RO 60 0175 was found to induce hypolocomotion in rats at doses greater than 0.5 mg/kg s.c., an effect reversed by the selective 5-HT(2C) receptor antagonist, SB-242084. RO 60 0175 did not elicit anxiolytic-like responses in the social interaction test under high light unfamiliar conditions, but suppressed both time spent in social interaction and locomotion at doses of 1 and 3 mg/kg s.c., suggesting a sedative response. In the Vogel conflict test, RO 60 0175 had no significant action on the number of shocks taken. In the Geller-Seifter test, RO 60 0175 (0.3 and 1 mg/kg s.c.) simultaneously reduced both unpunished and punished lever pressing, a profile consistent with sedation. Finally, RO 60 0175 was tested in a rat social interaction test under low light familiar conditions optimal for the detection of anxiogenic-like responses. At 1 and 3 mg/kg s.c., RO 60 0175 reduced both time spent in social interaction and concurrent locomotion, a profile more consistent with sedation than anxiogenesis. In conclusion, RO 60 0175 induced sedative-like responses via 5-HT(2C) receptor activation, but was neither anxiolytic, nor clearly anxiogenic at the doses tested. PMID:10650160

The serotonin 5-HT2A, 5-HT2B, and 5-HT2C G protein-coupled receptors signal primarily through Gαq to activate phospholipase C (PLC) and formation of inositol phosphates (IP) and diacylglycerol. The human 5-HT2Creceptor, expressed exclusively in the central nervous system, is involved in several physiological and psychological processes. Development of 5-HT2Cagonists that do not also activate 5-HT2A or 5-HT2B receptors is challenging because transmembrane domain identity is about 75% among 5-HT2 subtypes. This paper reports 5-HT2 receptor affinity and function of (1R,3S)-(−)-trans-1-phenyl-3-dimethylamino-1,2,3,4-tetrahydronaphthalene (PAT), a small molecule that produces anorexia and weight-loss after peripheral administration to mice. (−)-Trans-PAT is a stereoselective full-efficacy agonist at human 5-HT2Creceptors, plus, it is a 5-HT2A/5-HT2B inverse agonist and competitive antagonist. The Ki of (−)-trans-PAT at 5-HT2A, 5-HT2B, and 5-HT2Creceptors is 410, 1200, and 37 nM, respectively. Functional studies measured activation of PLC/[3H]-IP formation in clonal cells expressing human 5-HT2 receptors. At 5-HT2Creceptors, (−)-trans-PAT is an agonist (EC50 = 20 nM) comparable to serotonin in potency and efficacy. At 5-HT2A and 5-HT2B receptors, (−)-trans-PAT is an inverse agonist (IC50 = 490 and 1,000 nM, respectively) and competitive antagonist (KB = 460 and 1400 nM, respectively) of serotonin. Experimental results are interpreted in light of molecular modeling studies indicating the (−)-trans-PAT protonated amine can form an ionic bond with D3.32 of 5-HT2A and 5-HT2Creceptors, but, not with 5-HT2B receptors. In addition to probing 5-HT2 receptor structure and function, (−)-trans-PAT is a novel lead regarding 5-HT2Cagonist/5-HT2A inverse agonist drug development for obesity and neuropsychiatric disorders. PMID:19397907

Serotonergic systems in the brain have been found to be important in the addiction to alcohol. The purpose of this study was to evaluate the efficacy of a novel 5-HT2creceptoragonist, lorcaserin for reducing alcohol consumption in alcohol-preferring (P) rats. Adult female rats were allowed to drink water or alcohol (12%, v/v) using a standard two-bottle choice procedure. Once stable baselines were established, the acute (0, 0.3125, 0.625 and 1.25 mg/kg, s.c.), and chronic (0, 0.625 mg/kg, sc for 10 days) effects of lorcaserin on alcohol intake and preference were assessed at different time points. In a separate experiment, the effects of lorcaserin on locomotor activity were determined. Our results show that both 0.625 and 1.25 mg/kg lorcaserin significantly reduced alcohol intake at 2, 4 and 6 h. after the drug administration. The chronic administration of 0.625 mg/kg lorcaserin significantly reduced alcohol intake up to 6h every day after the injection and there was no sign of diminished efficacy of the drug during 10-day treatment. To determine the effects of lorcaserin on sucrose intake, rats were put on a two-bottle choice of water vs a solution of 7% sucrose. The high dose of lorcaserin (1.25 mg/kg, s.c.) reduced sucrose intake only for up to 2 h. When tested for locomotor activity, lorcaserin injected 20 min before testing significantly reduced locomotor activity at all doses. However, when it was injected 5.5h before the start of the 1-h session, neither dose had a significant effect on locomotor activity. These results show the efficacy of lorcaserin in reducing alcohol intake without a significant effect on water intake and locomotion suggesting the involvement of 5-HT2creceptors in alcohol seeking behavior. Further research is warranted to determine the possible efficacy of lorcaserin or similar drugs as treatments for the treatment of alcoholism. PMID:25109272

We report here the design, synthesis, and pharmacological properties of a series of compounds related to tranylcypromine (9), which itself was discovered as a lead compound in a high-throughput screening campaign. Starting from 9, which shows modest activity as a 5-HT2Cagonist, a series of 1-aminomethyl-2-phenylcyclopropanes was investigated as 5-HT2Cagonists through iterative structural modifications. Key pharmacophore feature of this new class of ligands is a 2-aminomethyl-trans-cyclopropyl side chain attached to a substituted benzene ring. Among the tested compounds, several were potent and efficacious 5-HT2Creceptoragonists with selectivity over both 5-HT2A and 5-HT2B receptors in functional assays. The most promising compound is 37 with 120- and 14-fold selectivity over 5-HT2A and 5-HT2B, respectively (EC50 = 585, 65, and 4.8 nM at the 2A, 2B, and 2C subtypes, respectively). In animal studies, compound 37 (10–60 mg/kg) decreased immobility time in the mouse forced swim test. PMID:19284718

Development of 5-HT2Cagonists for treatment of neuropsychiatric disorders, including psychoses, substance abuse, and obesity, has been fraught with difficulties, because the vast majority of reported 5-HT2C selective agonists also activate 5-HT2A and/or 5-HT2B receptors, potentially causing hallucinations and/or cardiac valvulopathy. Herein is described a novel, potent, and efficacious human 5-HT2Creceptoragonist, (-)-trans-(2S,4R)-4-(3'[meta]-bromophenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (-)-MBP), that is a competitive antagonist and inverse agonist at human 5-HT2A and 5-HT2B receptors, respectively. (-)-MBP has efficacy comparable to the prototypical second-generation antipsychotic drug clozapine in three C57Bl/6 mouse models of drug-induced psychoses: the head-twitch response elicited by [2,5]-dimethoxy-4-iodoamphetamine; hyperlocomotion induced by MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (dizocilpine maleate)]; and hyperlocomotion induced by amphetamine. (-)-MBP, however, does not alter locomotion when administered alone, distinguishing it from clozapine, which suppresses locomotion. Finally, consumption of highly palatable food by mice was not increased by (-)-MBP at a dose that produced at least 50% maximal efficacy in the psychoses models. Compared with (-)-MBP, the enantiomer (+)-MBP was much less active across in vitro affinity and functional assays using mouse and human receptors and also translated in vivo with comparably lower potency and efficacy. Results indicate a 5-HT2Creceptor-specific agonist, such as (-)-MBP, may be pharmacotherapeutic for psychoses, without liability for obesity, hallucinations, heart disease, sedation, or motoric disorders. PMID:24563531

Development of 5-HT2Cagonists for treatment of neuropsychiatric disorders, including psychoses, substance abuse, and obesity, has been fraught with difficulties, because the vast majority of reported 5-HT2C selective agonists also activate 5-HT2A and/or 5-HT2B receptors, potentially causing hallucinations and/or cardiac valvulopathy. Herein is described a novel, potent, and efficacious human 5-HT2Creceptoragonist, (−)-trans-(2S,4R)-4-(3′[meta]-bromophenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (−)-MBP), that is a competitive antagonist and inverse agonist at human 5-HT2A and 5-HT2B receptors, respectively. (−)-MBP has efficacy comparable to the prototypical second-generation antipsychotic drug clozapine in three C57Bl/6 mouse models of drug-induced psychoses: the head-twitch response elicited by [2,5]-dimethoxy-4-iodoamphetamine; hyperlocomotion induced by MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (dizocilpine maleate)]; and hyperlocomotion induced by amphetamine. (−)-MBP, however, does not alter locomotion when administered alone, distinguishing it from clozapine, which suppresses locomotion. Finally, consumption of highly palatable food by mice was not increased by (−)-MBP at a dose that produced at least 50% maximal efficacy in the psychoses models. Compared with (−)-MBP, the enantiomer (+)-MBP was much less active across in vitro affinity and functional assays using mouse and human receptors and also translated in vivo with comparably lower potency and efficacy. Results indicate a 5-HT2Creceptor-specific agonist, such as (−)-MBP, may be pharmacotherapeutic for psychoses, without liability for obesity, hallucinations, heart disease, sedation, or motoric disorders. PMID:24563531

To understand molecular determinants for ligand activation of the serotonin 5-HT2C G protein-coupled receptor (GPCR), a drug target for obesity and neuropsychiatric disorders, a 5-HT2C homology model was built according to an adrenergic β2 GPCR (β2AR) structure and validated using a 5-HT2B GPCR crystal structure. The models were equilibrated in a simulated phosphatidyl choline membrane for ligand docking and molecular dynamics studies. Ligands included (2S, 4R)-(-)-trans-4-(3'-bromo- and trifluoro-phenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalene-2-amine (3'-Br-PAT and 3'-CF3-PAT), a 5-HT2Cagonist and inverse agonist, respectively. Distinct interactions of 3'-Br-PAT and 3'-CF3-PAT at the wild-type (WT) 5-HT2Creceptor model were observed and experimental 5-HT2Creceptor mutagenesis studies were undertaken to validate the modelling results. For example, the inverse agonist 3'-CF3-PAT docked deeper in the WT 5-HT2C binding pocket and altered the orientation of transmembrane helices (TM) 6 in comparison to the agonist 3'-Br-PAT, suggesting that changes in TM orientation that result from ligand binding impact function. For both PATs, mutation of 5-HT2C residues S3.36, T3.37, and F5.47 to alanine resulted in significantly decreased affinity, as predicted from modelling results. It was concluded that upon PAT binding, 5-HT2C residues T3.37 and F5.47 in TMs 3 and 5, respectively, engage in inter-helical interactions with TMs 4 and 6, respectively. The movement of TMs 5 and 6 upon agonist and inverse agonist ligand binding observed in the 5-HT2Creceptor modelling studies was similar to movements reported for the activation and deactivation of the β2AR, suggesting common mechanisms among aminergic neurotransmitter GPCRs.

Lorcaserin, a serotonin (5-hydroxytryptamine, 5-HT) 2Creceptoragonist, was recently approved for the treatment of obesity. We previously suggested that 5-HT2Creceptoragonists affect reward processes and reduce the rewarding effects of drugs of abuse. Here, we determined whether lorcaserin (1) decreases responding for brain stimulation reward (BSR) and (2) prevents nicotine from enhancing the efficacy of BSR. Rats were trained on the intracranial self-stimulation (ICSS) paradigm to nosepoke for BSR of either the dorsal raphé nucleus or left medial forebrain bundle. In Experiment 1, lorcaserin (0.3-1.0 mg/kg) dose-dependently reduced the efficacy of BSR. This effect was blocked by prior administration of the 5-HT2Creceptor antagonist SB242084. In Experiment 2, separate groups of rats received saline or nicotine (0.4 mg/kg) for eight sessions prior to testing. Although thresholds were unaltered in saline-treated rats, nicotine reduced reward thresholds. An injection of lorcaserin (0.3 mg/kg) prior to nicotine prevented the reward-enhancing effect of nicotine across multiple test sessions. These results demonstrated that lorcaserin reduces the rewarding value of BSR and also prevents nicotine from facilitating ICSS. Hence, lorcaserin may be effective in treating psychiatric disorders, including obesity and nicotine addiction, by reducing the value of food or drug rewards. PMID:25781911

Lorcaserin (Lorqess, Belviq(®)) is a selective 5-HT(2C) receptoragonist that has received FDA approval for the treatment of obesity. 5-HT(2C) receptoragonists are also efficacious in decreasing multiple aspects of cocaine motivation and reward in preclinical models. This would suggest that lorcaserin is a clinically available therapeutic with the potential to treat cocaine addiction. Here we report the effects of lorcaserin (0.1 mg/kg-1.0 mg/kg) on multiple aspects of cocaine-related behaviours in rats. We find that lorcaserin dose-dependently decreases cocaine self-administration on progressive and fixed ratio schedules of reinforcement. Lorcaserin also reduces reinstatement of cocaine-seeking behaviour in response to priming injections of cocaine and/or reintroduction of cocaine-associated cues. Finally, lorcaserin dose-dependently decreases cocaine-induced hyperlocomotion. Our results, when considered in concert with similar emergent findings in non-human primates, strongly support continued research into the potential of lorcaserin as a clinical treatment for cocaine addiction. PMID:26427596

Serotonin 2Creceptor (5-HT2CR) agonists attenuate reinstatement of cocaine-seeking behavior. These receptors are found throughout the limbic system, including the basolateral amygdala (BlA), which is involved in forming associations between emotional stimuli and environmental cues, and the central amygdala (CeA), which is implicated in the expression of conditioned responding to emotional stimuli. This study investigated whether 5-HT2CRs in the amygdala are involved in cue and cocaine-primed reinstatement of cocaine-seeking behavior. Rats were trained to self-administer cocaine (0.75 mg/kg, i.v.) which that was paired with light and tone cues, and then subsequently they underwent daily extinction training. Rats then received bilateral microinfusions of the 5-HT2CR agonist CP809101 (0.01-1.0 μg/0.2 μl/side) into either the BlA or CeA prior to tests for cue or cocaine-primed (10 mg/kg, i.p.) reinstatement. Rats were also tested for CP809101 effects on anxiety-like behavior on the elevated plus-maze (EPM). Surprisingly, intra-BlA CP809101 had no effect on cue reinstatement, though it did increase anxiety-like behavior on the EPM. Intra-CeA infusions of CP809101 attenuated cocaine-primed reinstatement, an effect that was prevented with concurrent administration of the 5-HT2CR antagonist SB242084 (0.1 μg/0.2 μl/side). CP809101 had no effect on cue reinstatement or anxiety-like behavior on the EPM. These findings suggest that 5-HT2CRs in the BlA modulate anxiety, whereas those in the CeA modulate incentive motivational effects induced by cocaine priming injections. PMID:24984080

The 5-HT2Creceptoragonist lorcaserin (Belviq®) has been Food and Drug Administration (FDA) approved for the treatment of obesity. The present study is a back translational investigation into the effect of 28-day lorcaserin treatment in a diet-induced obesity (DIO) model using male, Sprague–Dawley rats. An assessment of drug effect on efficacy and multiple safety endpoints including cardiac function was undertaken. Lorcaserin (1–2 mg/kg SC b.i.d.) significantly reduced percentage body weight gain compared to vehicle-treated controls (VEH: 10.6 ± 0.4%; LOR 1: 7.6 ± 1.2%; LOR 2: 5.4 ± 0.6%). Measurement of body composition using quantitative magnetic resonance (QMR) imaging indicated this change was due to the selective reduction in body fat mass. Modest effects on food intake were recorded. At the completion of the treatment phase, echocardiography revealed no evidence for valvulopathy, that is, no aortic or mitral valve regurgitation. The pharmacokinetics of the present treatment regimen was determined over a 7-day treatment period; plasma Cmin and Cmax were in the range 13–160 ng/mL (1 mg/kg b.i.d.) and 34–264 ng/mL (2 mg/kg b.i.d.) with no evidence for drug accumulation. In sum, these studies show an effect of lorcaserin in the DIO model, that in the context of the primary endpoint measure of % body weight change was similar to that reported clinically (i.e., 3.0–5.2% vs. 3.2%). The present studies highlight the translational value of obesity models such as DIO, and suggest that assuming consideration is paid to nonspecific drug effects such as malaise, the DIO model has reasonable forward translational value to help predict clinical outcomes of a new chemical entity. PMID:25692009

The 5-HT2Creceptoragonist lorcaserin (Belviq®) has been Food and Drug Administration (FDA) approved for the treatment of obesity. The present study is a back translational investigation into the effect of 28-day lorcaserin treatment in a diet-induced obesity (DIO) model using male, Sprague-Dawley rats. An assessment of drug effect on efficacy and multiple safety endpoints including cardiac function was undertaken. Lorcaserin (1-2 mg/kg SC b.i.d.) significantly reduced percentage body weight gain compared to vehicle-treated controls (VEH: 10.6 ± 0.4%; LOR 1: 7.6 ± 1.2%; LOR 2: 5.4 ± 0.6%). Measurement of body composition using quantitative magnetic resonance (QMR) imaging indicated this change was due to the selective reduction in body fat mass. Modest effects on food intake were recorded. At the completion of the treatment phase, echocardiography revealed no evidence for valvulopathy, that is, no aortic or mitral valve regurgitation. The pharmacokinetics of the present treatment regimen was determined over a 7-day treatment period; plasma C min and C max were in the range 13-160 ng/mL (1 mg/kg b.i.d.) and 34-264 ng/mL (2 mg/kg b.i.d.) with no evidence for drug accumulation. In sum, these studies show an effect of lorcaserin in the DIO model, that in the context of the primary endpoint measure of % body weight change was similar to that reported clinically (i.e., 3.0-5.2% vs. 3.2%). The present studies highlight the translational value of obesity models such as DIO, and suggest that assuming consideration is paid to nonspecific drug effects such as malaise, the DIO model has reasonable forward translational value to help predict clinical outcomes of a new chemical entity. PMID:25692009

The antidepressants currently used need improvement, especially in terms of efficacy, relapse rate and onset of action. In this review the clinical and experimental data which support the rationale for 5-HT2Cagonists in the treatment of depression are listed. Next, the results obtained with the non-selective 5-HT2Cagonists on the market and in clinical development are described. Finally, the preclinical data on the more selective 5-HT2Cagonists are summarized. These recent preclinical results reveal a greater potency and effect size compared to fluoxetine, good tolerability and no evidence of tolerance development. Selective 5-HT2Cagonists might become innovative drugs for the treatment of depression, panic, obsessive-compulsive disorder (OCD), some forms of aggression and eating disorders. PMID:16160946

The pharmaceutical compound, dihydroergotamine (DHE) is dispensed to prevent and reduce the occurrence of migraine attacks. Although still controversial, the prophylactic effect of this drug is believed to be caused through blockade and/or activation of numerous receptors including serotonin (5-HT) receptors of the 5-HT2 subtype. To elucidate if 5-HT2 receptors (5-HT2Rs) may be involved in DHE prophylactic effect, we performed investigations aimed to determine the respective pharmacological profile of DHE and of its major metabolite 8′-hydroxy-DHE (8′-OH-DHE) at the 5-HT2B and 5-HT2CRs by binding, inositol triphosphate (IP3) or cyclic GMP (cGMP) coupling studies in transfected fibroblasts. DHE and 8′-OH-DHE are competitive compounds at 5-HT2B and 5-HT2CRs. 8′-OH-DHE interaction at (5-HT2BRs) was best fitted by a biphasic competition curve and displayed the highest affinity with a Ki of 5 nM. These two compounds acted as agonists for both receptors in respect to cGMP production with pEC50 of 8.32±0.09 for 8′-OH-DHE at 5-HT2B and 7.83±0.06 at 5-HT2CRs. Knowing that the antimigraine prophylactic effect of DHE is only observed after long-term treatment, we chronically exposed the recombinant cells to DHE and 8′-OH-DHE. The number of 5-HT2BR-binding sites was always more affected than 5-HT2CRs. At 5-HT2BRs, 8′-OH-DHE was more effective than DHE, with an uncoupling that persisted for more than 40 h for IP3 or cGMP. By contrast, the 5-HT2CR coupling was reversible after either treatment. Chronic exposure to 8′-OH-DHE caused a persistent agonist-mediated desensitisation of 5-HT2B, but not 5-HT2CRs. This may be of relevance to therapeutic actions of the compound. PMID:12970106

This review provides an overview of the role of central serotonin2C (5-HT2C) receptors in drug addiction, specifically focusing on their impact on the neurochemical and behavioral effects of cocaine, one of the most worldwide abused drug. First, we described the neurochemical and electrophysiological mechanisms underlying the interaction between 5-HT2Creceptors and the mesocorticolimbic dopaminergic network, in keeping with the key role of this system in drug abuse and dependence. Thereafter, we focused on the role of 5-HT2Creceptors in the effects of cocaine in various preclinical behavioral models used in drug addiction research, such as locomotor hyperactivity, locomotor sensitization, drug discrimination, and self-administration, to end with an overview of the neurochemical mechanisms underlying the interactions between 5-HT2Creceptors, mesocorticolimbic dopamine system, and cocaine. On their whole, the presented data provide compelling preclinical evidence that 5-HT2Creceptoragonists may have efficacy in the treatment of cocaine abuse and dependence, thereby underlying the need for additional clinical studies to ascertain whether preclinical data translate to the human. PMID:23748692

The discovery of a new class of nonprostanoid prostaglandin I2 receptor (IP receptor) agonists is reported. Among them, the unique piperidine derivative 31b (2-((1-(2-(N-(4-tolyl)benzamido)ethyl)piperidin-4-yl)oxy)acetic acid) was a good IP receptoragonist and was 50-fold more selective for the human IP receptor than for other human prostanoid receptors. This compound showed good pharmacokinetic properties in dog. PMID:26996371

Opioid receptors, especially Kappa opioid receptor (KOR) play an important role in the pathophysiological process of cerebral ischemia reperfusion injury. Previously accepted KOR agonists activity has included anti-nociception, cardiovascular, anti-pruritic, diuretic, and antitussive effects, while compelling evidence from various ischemic animal models indicate that KOR agonist have neuroprotective effects through various mechanisms. In this review, we aimed to demonstrate the property of KOR agonist and its role in global and focal cerebral ischemia. Based on current preclinical research, the KOR agonists may be useful as a neuroprotective agent. The recent discovery of salvinorin A, highly selective non-opioid KOR agonist, offers a new tool to study the role of KOR in brain HI injury and the protective effects of KOR agonist. The unique pharmacological profile of salvinorin A along with the long history of human usage provides its high candidacy as a potential alternative medication for brain HI injury. PMID:25574482

N-Benzyl substitution of 5-HT2A receptoragonists of the phenethylamine structural class of psychedelics (such as 4-bromo-2,5-dimethoxyphenethylamine, often referred to as 2C-B) confer a significant increase in binding affinity as well as functional activity of the receptor. We have prepared a series of 48 compounds with structural variations in both the phenethylamine and N-benzyl part of the molecule to determine the effects on receptor binding affinity and functional activity at 5-HT2A and 5-HT2Creceptors. The compounds generally had high affinity for the 5-HT2A receptor with 8b having the highest affinity at 0.29 nM but with several other compounds also exhibiting subnanomolar binding affinities. The functional activity of the compounds was distributed over a wider range with 1b being the most potent at 0.074 nM. Most of the compounds exhibited low to moderate selectivity (1- to 40-fold) for the 5-HT2A receptor in the binding assays, although one compound 6b showed an impressive 100-fold selectivity for the 5-HT2A receptor. In the functional assay, selectivity was generally higher with 1b being more than 400-fold selective for the 5-HT2A receptor. PMID:24397362

The neuromodulator serotonin has a complex set of effects on the auditory responses of neurons within the inferior colliculus (IC), a midbrain auditory nucleus that integrates a wide range of inputs from auditory and nonauditory sources. To determine whether activation of different types of serotonin receptors is a source of the variability in serotonergic effects, four selective agonists of serotonin receptors in the serotonin (5-HT) 1 and 5-HT2 families were iontophoretically applied to IC neurons, which were monitored for changes in their responses to auditory stimuli. Different agonists had different effects on neural responses. The 5-HT1A agonist had mixed facilitatory and depressive effects, whereas 5-HT1B and 5-HT2Cagonists were both largely facilitatory. Different agonists changed threshold and frequency tuning in ways that reflected their effects on spike count. When pairs of agonists were applied sequentially to the same neurons, selective agonists sometimes affected neurons in ways that were similar to serotonin, but not to other selective agonists tested. Different agonists also differentially affected groups of neurons classified by the shapes of their frequency-tuning curves, with serotonin and the 5-HT1 receptors affecting proportionally more non-V-type neurons relative to the other agonists tested. In all, evidence suggests that the diversity of serotonin receptor subtypes in the IC is likely to account for at least some of the variability of the effects of serotonin and that receptor subtypes fulfill specialized roles in auditory processing. PMID:16870843

In two inbred strains of mice, C57BL/6 and 129Sv, the majority of forebrain neocortical pre-mRNA encoding the serotonin 2C (5-HT2C) receptor is altered by adenosine-to-inosine editing. As a result, >60% of all mRNAs encode receptors with reduced constitutive and agonist-stimulated activity. However, in the BALB/c strain, a genetically distinct inbred strain with lower forebrain serotonin levels, spontaneously elevated anxiety, and increased stress reactivity, the majority of 5-HT2C mRNA is nonedited and encodes receptors with the highest constitutive activity and the highest agonist affinity and potency. Neither acute stress (the forced swim test) nor chronic treatment with the serotonin-selective reuptake inhibitor fluoxetine elicit significant changes in 5-HT2C pre-mRNA editing in C57BL/6 mice. In contrast, exposure of BALB/c mice to acute stress and chronic treatment of nonstressed BALB/c mice with fluoxetine elicit significant, site-specific increases in 5-HT2C pre-mRNA editing that increase the pool of mRNA encoding receptors with reduced function. These changes in 5-HT2C pre-mRNA editing resemble those detected previously in the prefrontal cortex of subjects with major depression. However, when chronic fluoxetine treatment is combined with stress exposure of BALB/c mice, these changes in 5-HT2C pre-mRNA editing are no longer detected. These findings illustrate that 5-HT2C pre-mRNA editing responses to stress and chronic fluoxetine are modulated by the genetic background, as well as the behavioral state of the animal. They suggest further that the changes in 5-HT2C pre-mRNA editing found in major depression reflect a previously unrecognized molecular response to stress that can be prevented by chronic antidepressant treatment. PMID:15659601

Agomelatine (AGM), an analog of melatonin, is a potential agonist at melatonin receptors 1/2 and a selective antagonist at 5-hydroxytryptamine 2Creceptors. AGM is widely used for the treatment of major depressive episodes in adults. However, multiple adverse effects associated with AGM have been re...

Understanding the intricate pathways that modulate appetite and subsequent food intake is of particular importance considering the rise in the incidence of obesity across the globe. The serotonergic system, specifically the 5-HT2Creceptor, has been shown to be of critical importance in the regulation of appetite and satiety. The GHS-R1a receptor is another key receptor that is well-known for its role in the homeostatic control of food intake and energy balance. We recently showed compelling evidence for an interaction between the GHS-R1a receptor and the 5-HT2Creceptor in an in vitro cell line system heterologously expressing both receptors. Here, we investigated this interaction further. First, we show that the GHS-R1a/5-HT2C dimer-induced attenuation of calcium signaling is not due to coupling to GαS, as no increase in cAMP signaling is observed. Next, flow cytometry fluorescence resonance energy transfer (fcFRET) is used to further demonstrate the direct interaction between the GHS-R1a receptor and 5-HT2Creceptor. In addition, we demonstrate colocalized expression of the 5-HT2C and GHS-R1a receptor in cultured primary hypothalamic and hippocampal rat neurons, supporting the biological relevance of a physiological interaction. Furthermore, we demonstrate that when 5-HT2Creceptor signaling is blocked ghrelin's orexigenic effect is potentiated in vivo. In contrast, the specific 5-HT2Creceptoragonist lorcaserin, recently approved for the treatment of obesity, attenuates ghrelin-induced food intake. This underscores the biological significance of our in vitro findings of 5-HT2Creceptor-mediated attenuation of GHS-R1a receptor activity. Together, this study demonstrates, for the first time, that the GHS-R1a/5-HT2Creceptor interaction translates into a biologically significant modulation of ghrelin's orexigenic effect. This data highlights the potential development of a combined GHS-R1a and 5-HT2Creceptor treatment strategy in weight management. PMID:25727097

Membranous nephropathy is one of the most common causes of nephrotic syndrome in adults. Recent reports suggest that treatment with adrenocorticotropic hormone (ACTH) reduces proteinuria, but the mechanism of action is unknown. Here, we identified gene expression of the melanocortin receptor MC1R in podocytes, glomerular endothelial cells, mesangial cells, and tubular epithelial cells. Podocytes expressed most MC1R protein, which colocalized with synaptopodin but not with an endothelial-specific lectin. We treated rats with passive Heymann nephritis (PHN) with MS05, a specific MC1R agonist, which significantly reduced proteinuria compared with untreated PHN rats (P < 0.01). Furthermore, treatment with MC1R agonists improved podocyte morphology and reduced oxidative stress. In summary, podocytes express MC1R, and MC1R agonism reduces proteinuria, improves glomerular morphology, and reduces oxidative stress in nephrotic rats with PHN. These data may explain the proteinuria-reducing effects of ACTH observed in patients with membranous nephropathy, and MC1R agonists may provide a new therapeutic option for these patients. PMID:20507942

Serotonin 2Creceptor (5-HT(2C)R) agonists administered systemically attenuate both cocaine-primed and cue-elicited reinstatement of extinguished cocaine-seeking behavior. To further elucidate the function of these receptors in addiction-like processes, this study examined the effects of microinfusing the 5-HT(2C)R agonist MK212 (0, 10, 30, 100 ng/side/0.2 microl) into the medial prefrontal cortex (mPFC) on cocaine self-administration and reinstatement of extinguished cocaine-seeking behavior. Male Sprague-Dawley rats were trained to self-administer cocaine (0.75 mg/kg, i.v.) paired with light and tone cues. Once responding stabilized, rats received MK212 microinfusions before tests for maintenance of cocaine self-administration. Next, extinction training to reduce cocaine-seeking behavior, defined as responses performed without cocaine reinforcement available, occurred until low extinction baselines were achieved. Rats then received MK212 microinfusions before tests for reinstatement of extinguished cocaine-seeking behavior elicited by cocaine-priming injections (10 mg/kg, i.p.) or response-contingent presentations of the cocaine-associated cues; operant responses during cocaine-primed reinstatement tests produced no consequences. MK212 microinfusions into the prelimbic and infralimbic, but not anterior cingulate, regions of the mPFC dose-dependently attenuated both cocaine-primed and cue-elicited reinstatement of extinguished cocaine-seeking behavior, but did not reliably affect cocaine self-administration. A subsequent experiment showed that the effects of MK212 (100 ng/side/0.2 microl) on reinstatement of extinguished cocaine-seeking behavior were blocked by co-administration of the 5-HT(2C)R antagonist SB242084 (200 ng/side/0.2 microl). MK212 administered alone into the mPFC as a drug prime produced no discernable effects on cocaine-seeking behavior. These findings suggest that stimulation of 5-HT(2C)Rs in the mPFC attenuates the incentive motivational effects

Recent work has identified nucleotide agonists selective for P2Y1, P2Y2 and P2Y6 receptors and nucleotide antagonists selective for P2Y1, P2Y12 and P2X1 receptors. Selective non-nucleotide antagonists have been reported for P2Y1, P2Y2, P2Y6, P2Y12, P2Y13, P2X2/3/P2X3 and P2X7 receptors. For example, the dinucleotide INS 37217 (Up4dC) potently activates the P2Y2 receptor, and the non-nucleotide antagonist A-317491 is selective for P2X2/3/P2X3 receptors. Nucleotide analogues in which the ribose moiety is substituted by a variety of novel ring systems, including conformation-ally locked moieties, have been synthesized as ligands for P2Y receptors. The focus on conformational factors of the ribose-like moiety allows the inclusion of general modifications that lead to enhanced potency and selectivity. At P2Y1,2,4,11 receptors, there is a preference for the North conformation as indicated with (N)-methanocarba analogues. The P2Y1 antagonist MRS2500 inhibited ADP-induced human platelet aggregation with an IC50 of 0.95 nM. MRS2365, an (N)-methanocarba analogue of 2-MeSADP, displayed potency (EC50) of 0.4 nM at the P2Y1 receptor, with >10 000-fold selectivity in comparison to P2Y12 and P2Y13 receptors. At P2Y6 receptors there is a dramatic preference for the South conformation. Three-dimensional structures of P2Y receptors have been deduced from structure activity relationships (SAR), mutagenesis and modelling studies. Detailed three-dimensional structures of P2X receptors have not yet been proposed. PMID:16805423

Cannabinoid receptoragonists such as delta-9-tetrahydrocannabinol (Δ(9)-THC) enhance some (antinociceptive) but not other (positive reinforcing) effects of mu opioid receptoragonists, suggesting that cannabinoids might be combined with opioids to treat pain without increasing, and possibly decreasing, abuse. The degree to which cannabinoids enhance antinociceptive effects of opioids varies across drugs insofar as Δ(9)-THC and the synthetic cannabinoid receptoragonist CP55940 increase the potency of some mu opioid receptoragonists (e.g., fentanyl) more than others (e.g., nalbuphine). It is not known whether interactions between cannabinoids and opioids vary similarly for other (abuse-related) effects. This study examined whether Δ(9)-THC and CP55940 differentially impact the discriminative stimulus effects of fentanyl and nalbuphine in monkeys (n=4) discriminating 0.01mg/kg of fentanyl (s.c.) from saline. Fentanyl (0.00178-0.0178mg/kg) and nalbuphine (0.01-0.32mg/kg) dose-dependently increased drug-lever responding. Neither Δ(9)-THC (0.032-1.0mg/kg) nor CP55940 (0.0032-0.032mg/kg) enhanced the discriminative stimulus effects of fentanyl or nalbuphine; however, doses of Δ(9)-THC and CP55940 that shifted the nalbuphine dose-effect curve markedly to the right and/or down were less effective or ineffective in shifting the fentanyl dose-effect curve. The mu opioid receptor antagonist naltrexone (0.032mg/kg) attenuated the discriminative stimulus effects of fentanyl and nalbuphine similarly. These data indicate that the discriminative stimulus effects of nalbuphine are more sensitive to attenuation by cannabinoids than those of fentanyl. That the discriminative stimulus effects of some opioids are more susceptible to modification by drugs from other classes has implications for developing maximally effective therapeutic drug mixtures with reduced abuse liability. PMID:27184925

Neurotransmitter receptors that control the release of opioid peptides in the spinal cord may play an important role in pain modulation. Norepinephrine, released by a descending pathway originating in the brainstem, is a powerful inducer of analgesia in the spinal cord. Adrenergic α2Creceptors are present in opioid-containing terminals in the dorsal horn, where they could modulate opioid release. The goal of this study was to investigate this possibility. Opioid release was evoked from rat spinal cord slices by incubating them with the sodium channel opener veratridine in the presence of peptidase inhibitors (actinonin, captopril and thiorphan), and was measured in situ through the internalization of μ-opioid receptors in dorsal horn neurons. Veratridine produced internalization in 70% of these neurons. The α2 receptoragonists clonidine, guanfacine, medetomidine and UK-14304 inhibited the evoked μ-opioid receptor internalization with IC50s of 1.7 μM, 248 nM, 0.3 nM and 22 nM, respectively. However, inhibition by medetomidine was only partial, and inhibition by UK-14304 reversed itself at concentrations higher than 50 nM. None of these agonists inhibited μ-opioid receptor internalization produced by endomorphin-2, showing that they inhibited opioid release and not the internalization itself. The inhibition produced by clonidine, guanfacine or UK-14304 was completely reversed by the selective α2C antagonist JP-1203. In contrast, inhibition by guanfacine was not prevented by the α2A antagonist BRL-44408. These results show that α2Creceptors inhibit the release of opioids in the dorsal horn. This action may serve to shut down the opioid system when the adrenergic system is active. PMID:18343461

Neurobehavioral investigations into the functions of estrogen receptor (ER)alpha and ERbeta have utilized 'knockout' mice, phytoestrogens and, more recently, ER-specific agonists. Feeding, sexual, aggressive and social behavior, anxiety, depression, drug abuse, pain perception, and learning (and associated synaptic plasticity) are affected by ERalpha and ERbeta in a manner that is dependent upon the specific behavior studied, gender and developmental stage. Overall, ERalpha and ERbeta appear to function together to foster sociosexual behavior while inhibiting behaviors that, if occurring at the time of behavioral estrous, may compete with reproduction (eg, feeding). Recently developed pharmacological tools have limited selectivity and availability to the research community at large, as they are not commercially available. The development of highly selective, commercially available ERbeta-specific antagonists would greatly benefit preclinical and applied research. PMID:18600582

Because of proven efficacy, reduced side effects, and less concern about addiction, non-benzodiazepine receptoragonists (non-BzRA) have become the most commonly prescribed hypnotic agents to treat onset and maintenance insomnia. First-line treatment is cognitive-behavioral therapy. When pharmacologic treatment is indicated, non-BzRA are first-line agents for the short-term and long-term management of transient and chronic insomnia related to adjustment, psychophysiologic, primary, and secondary causation. In this article, the benefits and risks of non-BzRA are reviewed, and the selection of a hypnotic agent is defined, based on efficacy, pharmacologic profile, and adverse events. PMID:26055674

The dynamic interplay between serotonin [5-hydroxytryptamine (5-HT)] neurotransmission and the hypothalamic-pituitary-adrenal (HPA) axis has been extensively studied over the past 30 years, but the underlying mechanism of this interaction has not been defined. A possibility receiving little attention is that 5-HT regulates upstream corticotropin-releasing hormone (CRH) signaling systems via activation of serotonin 2Creceptors (5-HT(2C)Rs) in the paraventricular nucleus of the hypothalamus (PVH). Through complementary approaches in wild-type rodents and 5-HT(2C)R-deficient mice, we determined that 5-HT(2C)Rs are necessary for 5-HT-induced HPA axis activation. We used laser-capture PVH microdissection followed by microarray analysis to compare the expression of 13 5-HTRs. Only 5-HT(2C)R and 5-HT(1D)R transcripts were consistently identified as present in the PVH, and of these, the 5-HT(2C)R was expressed at a substantially higher level. The abundant expression of 5-HT(2C)Rs in the PVH was confirmed with in situ hybridization histochemistry. Dual-neurohistochemical labeling revealed that approximately one-half of PVH CRH-containing neurons coexpressed 5-HT(2C)R mRNA. We observed that PVH CRH neurons consistently depolarized in the presence of a high-affinity 5-HT(2C)R agonist, an effect blocked by a 5-HT(2C)R antagonist. Supporting the importance of 5-HT(2C)Rs in CRH neuronal activity, genetic inactivation of 5-HT(2C)Rs produced a downregulation of CRH mRNA and blunted CRH and corticosterone release after 5-HT compound administration. These findings thus provide a mechanistic explanation for the longstanding observation of HPA axis stimulation in response to 5-HT and thereby give insight into the neural circuitry mediating the complex neuroendocrine responses to stress. PMID:17596444

G protein-coupled receptors (GPCRs) are a prominent class of plasma membrane proteins that regulate physiologic responses to a wide variety of stimuli and therapeutic agents. Although GPCR oligomerization has been studied extensively in recombinant cells, it remains uncertain whether native receptors expressed in their natural cellular environment are monomers, dimers, or oligomers. The goal of this study was to determine the monomer/oligomer status of a native GPCR endogenously expressed in its natural cellular environment. Native 5-HT2Creceptors in choroid plexus epithelial cells were evaluated using fluorescence correlation spectroscopy (FCS) with photon counting histogram (PCH). An anti-5-HT2C fragment antigen binding protein was used to label native 5-HT2Creceptors. A known monomeric receptor (CD-86) served as a control for decoding the oligomer status of native 5-HT2Creceptors by molecular brightness analysis. FCS with PCH revealed molecular brightness values for native 5-HT2Creceptors equivalent to the molecular brightness of a homodimer. 5-HT2Creceptors displayed a diffusion coefficient of 5 × 10(-9) cm(2)/s and were expressed at 32 receptors/μm(2) on the apical surface of choroid plexus epithelial cells. The functional significance and signaling capabilities of the homodimer were investigated in human embryonic kidney 293 cells using agonists that bind in a wash-resistant manner to one or both protomers of the homodimer. Whereas agonist binding to one protomer resulted in G protein activation, maximal stimulation required occupancy of both protomers. This study is the first to demonstrate the homodimeric structure of 5-HT2Creceptors endogenously expressed in their native cellular environment, and identifies the homodimer as a functional signaling unit. PMID:25609374

G protein–coupled receptors (GPCRs) are a prominent class of plasma membrane proteins that regulate physiologic responses to a wide variety of stimuli and therapeutic agents. Although GPCR oligomerization has been studied extensively in recombinant cells, it remains uncertain whether native receptors expressed in their natural cellular environment are monomers, dimers, or oligomers. The goal of this study was to determine the monomer/oligomer status of a native GPCR endogenously expressed in its natural cellular environment. Native 5-HT2Creceptors in choroid plexus epithelial cells were evaluated using fluorescence correlation spectroscopy (FCS) with photon counting histogram (PCH). An anti–5-HT2C fragment antigen binding protein was used to label native 5-HT2Creceptors. A known monomeric receptor (CD-86) served as a control for decoding the oligomer status of native 5-HT2Creceptors by molecular brightness analysis. FCS with PCH revealed molecular brightness values for native 5-HT2Creceptors equivalent to the molecular brightness of a homodimer. 5-HT2Creceptors displayed a diffusion coefficient of 5 × 10−9 cm2/s and were expressed at 32 receptors/μm2 on the apical surface of choroid plexus epithelial cells. The functional significance and signaling capabilities of the homodimer were investigated in human embryonic kidney 293 cells using agonists that bind in a wash-resistant manner to one or both protomers of the homodimer. Whereas agonist binding to one protomer resulted in G protein activation, maximal stimulation required occupancy of both protomers. This study is the first to demonstrate the homodimeric structure of 5-HT2Creceptors endogenously expressed in their native cellular environment, and identifies the homodimer as a functional signaling unit. PMID:25609374

Although G protein-coupled receptors (GPCRs) are traditionally categorized as Gs-, Gq-, or Gi/o-coupled, their signaling is regulated by multiple mechanisms. GPCRs can couple to several effector pathways, having the capacity to interact not only with more than one G protein subtype but also with alternative signaling or effector proteins such as arrestins. Moreover, GPCR ligands can have different efficacies for activating these signaling pathways, a characteristic referred to as biased agonism or functional selectivity. In this work our aim was to detect differences in the ability of various agonists acting at the α2C type of adrenergic receptors (α2C-ARs) to modulate cAMP accumulation, cytoplasmic Ca(2+) release, β-arrestin recruitment and receptor internalization. A detailed comparative pharmacological characterization of G protein-dependent and -independent signaling pathways was carried out using adrenergic agonists (norepinephrine, phenylephrine, brimonidine, BHT-920, oxymetazoline, clonidine, moxonidine, guanabenz) and antagonists (MK912, yohimbine). As initial analysis of agonist Emax and EC50 values suggested possible functional selectivity, ligand bias was quantified by applying the relative activity scale and was compared to that of the endogenous agonist norepinephrine. Values significantly different from 0 between pathways indicated an agonist that promoted different level of activation of diverse effector pathways most likely due to the stabilization of a subtly different receptor conformation from that induced by norepinephrine. Our results showed that a series of agonists acting at the α2C-AR displayed different degree of functional selectivity (bias factors ranging from 1.6 to 36.7) through four signaling pathways. As signaling via these pathways seems to have distinct functional and physiological outcomes, studying all these stages of receptor activation could have further implications for the development of more selective therapeutics with

In acute organ injuries, mitochondria are often dysfunctional, and recent research has revealed that recovery of mitochondrial and renal functions is accelerated by induction of mitochondrial biogenesis (MB). We previously reported that the nonselective 5-HT2 receptoragonist DOI [1-(4-iodo-2,5-dimethoxyphenyl)propan-2-amine] induced MB in renal proximal tubular cells (RPTCs). The goal of this study was to determine the role of 5-HT2 receptors in the regulation of mitochondrial genes and oxidative metabolism in the kidney. The 5-HT2Creceptoragonist CP-809,101 [2-[(3-chlorophenyl)methoxy]-6-(1-piperazinyl)pyrazine] and antagonist SB-242,084 [6-chloro-2,3-dihydro-5-methyl-N-[6-[(2-methyl-3-pyridinyl)oxy]-3-pyridinyl]-1H-indole-1-carboxyamide dihydrochloride] were used to examine the induction of renal mitochondrial genes and oxidative metabolism in RPTCs and in mouse kidneys in the presence and absence of the 5-HT2Creceptor. Unexpectedly, both CP-809,101 and SB-242,084 increased RPTC respiration and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) mRNA expression in RPTCs at 1-10 nM. In addition, CP-809,101 and SB-242,084 increased mRNA expression of PGC-1α and the mitochondrial proteins NADH dehydrogenase subunit 1 and NADH dehydrogenase (ubiquinone) β subcomplex 8 in mice. These compounds increased mitochondrial genes in RPTCs in which the 5-HT2Creceptor was downregulated with small interfering RNA and in the renal cortex of mice lacking the 5-HT2Creceptor. By contrast, the ability of these compounds to increase PGC-1α mRNA and respiration was blocked in RPTCs treated with 5-HT2A receptor small interfering RNA or the 5-HT2A receptor antagonist eplivanserin. In addition, the 5-HT2A receptoragonist NBOH-2C-CN [4-[2-[[(2-hydroxyphenyl)methyl]amino]ethyl]-2,5-dimethoxybenzonitrile] increased RPTC respiration at 1-100 nM. These results suggest that agonism of the 5-HT2A receptor induces MB and that the classic 5-HT2Creceptoragonist CP

1. The Drosophila melanogaster gamma-aminobutyric acid (GABA) receptor subunits, RDLac and DRC 17-1-2, form functional homo-oligomeric receptors when heterologously expressed in Xenopus laevis oocytes. The subunits differ in only 17 amino acids, principally in regions of the N-terminal domain which determine agonist pharmacology in vertebrate ionotropic neurotransmitter receptors. A range of conformationally restricted GABA analogues were tested on the two homo-oligomers and their agonists pharmacology compared with that of insect and vertebrate iontropic GABA receptors. 2. The actions of GABA, isoguvacine and isonipecotic acid on RDLac and DRC 17-1-2 homo-oligomers were compared, by use of two-electrode voltage-clamp. All three compounds were full agonists of both receptors, but were 4-6 fold less potent agonists of DRC 17-1-2 homo-oligomers than of RDLac. However, the relative potencies of these agonists on each receptor were very similar. 3. A more complete agonist profile was established for RDLac homo-oligomers. The most potent agonists of these receptors were GABA, muscimol and trans-aminocrotonic acid (TACA), which were approximately equipotent. RDLac homo-oligomers were fully activated by a range of GABA analogues, with the order of potency: GABA > ZAPA ((Z)-3-[(aminoiminomethyl)thio]prop-2-enoic acid) > isoguvacine > imidazole-4-acetic acid > or = isonipecotic acid > or = cis-aminocrotonic acid (CACA) > beta-alanine. 3-Aminopropane sulphonic acid (3-APS), a partial agonist of RDLac homo-oligomers, was the weakest agonist tested and 100 fold less potent than GABA. 4. SR95531, an antagonist of vertebrate GABAA receptors, competitively inhibited the GABA responses of RDLac homo-oligomers, which have previously been found to insensitive to bicuculline. However, its potency (IC50 500 microM) was much reduced when compared to GABAA receptors. 5. The agonist pharmacology of Drosophila RDLac homo-oligomers exhibits aspects of the characteristic pharmacology of

The serotonin subtype 2C (5HT2C) receptor is an emerging and promising drug target to treat several disorders of the human central nervous system. In this current report, two potent and selective 5HT2C full agonists, WAY-163909 (2) and vabicaserin (3), were radiolabeled with carbon-11 via Pictet–Spengler cyclization with [11C]formaldehyde and used in positron emission tomography (PET) imaging. Reaction conditions were optimized to exclude the major source of isotope dilution caused by the previously unknown breakdown of N,N-dimethylformamide (DMF) to formaldehyde at high temperature under mildly acid conditions. In vivo PET imaging was utilized to evaluate the pharmacokinetics and distribution of the carbon-11 labeled 5HT2Cagonists. Both radiolabeled molecules exhibit high blood–brain barrier (BBB) penetration and nonspecific binding, which was unaltered by preadministration of the unlabeled agonist. Our work demonstrates that Pictet–Spengler cyclization can be used to label drugs with carbon-11 to study their pharmacokinetics and for evaluation as PET radiotracers. PMID:24491146

Glucagon-like peptide-1 (GLP-1) receptoragonists are a new group of drugs for the treatment of type 2 diabetes mellitus (DM2). In the present article, we review the available evidence on the efficacy of GLP-1 receptoragonists as glucose-lowering agents, their place in therapeutic algorithms, and the clinical factors associated with a favorable treatment response. Finally, we describe the clinical characteristics of patients who may benefit from these drugs. PMID:25326839

Glucagon-like peptide-1 (GLP-1) receptoragonists are a new group of drugs for the treatment of type 2 diabetes mellitus (DM2). In the present article, we review the available evidence on the efficacy of GLP-1 receptoragonists as glucose-lowering agents, their place in therapeutic algorithms, and the clinical factors associated with a favorable treatment response. Finally, we describe the clinical characteristics of patients who may benefit from these drugs. PMID:25437461

BACKGROUND AND PURPOSE The opioid receptor family comprises four structurally homologous but functionally distinct sub-groups, the μ (MOP), δ (DOP), κ (KOP) and nociceptin (NOP) receptors. As most opioid agonists are selective but not specific, a broad spectrum of behaviours due to activation of different opioid receptors is expected. In this study, we examine whether other opioid receptor systems influenced KOP-mediated antinociception. EXPERIMENTAL APPROACH We used a tail withdrawal assay in C57Bl/6 mice to assay the antinociceptive effect of systemically administered opioid agonists with varying selectivity at KOP receptors. Pharmacological and genetic approaches were used to analyse the interactions of the other opioid receptors in modulating KOP-mediated antinociception. KEY RESULTS Etorphine, a potent agonist at all four opioid receptors, was not anti-nociceptive in MOP knockout (KO) mice, although etorphine is an efficacious KOP receptoragonist and specific KOP receptoragonists remain analgesic in MOP KO mice. As KOP receptoragonists are aversive, we considered KOP-mediated antinociception might be a form of stress-induced analgesia that is blocked by the anxiolytic effects of DOP receptoragonists. In support of this hypothesis, pretreatment with the DOP antagonist, naltrindole (10 mg·kg−1), unmasked etorphine (3 mg·kg−1) antinociception in MOP KO mice. Further, in wild-type mice, KOP-mediated antinociception by systemic U50,488H (10 mg·kg−1) was blocked by pretreatment with the DOP agonist SNC80 (5 mg·kg−1) and diazepam (1 mg·kg−1). CONCLUSIONS AND IMPLICATIONS Systemic DOP receptoragonists blocked systemic KOP antinociception, and these results identify DOP receptoragonists as potential agents for reversing stress-driven addictive and depressive behaviours mediated through KOP receptor activation. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles

Several lines of evidence suggest that brain serotonergic functions, including behavioral and neurochemical responses to 5-HT{sub 2C} agonist, are abnormal in some individuals with alcoholism and aggressive behaviors. The aim of the present study was to identify coding sequence variants in the human 5-HT{sub 2C} receptor gene which may cause abnormal or variant function of this receptor. Using SSCP analysis, a non-conservative cys-ser substitution was found in the 5-HT{sub 2C} receptor (designated 5-HT{sub 2Ccys} and 5-HT{sub 2Cser}). The polymorphism was typed in CEPH families to genetically map the gene. To test for association of the variant to alcoholism, violent behavior and serotonin function, the 5-HT{sub 2C} genotypes of 151 non-related Finnish male alcoholic violent offenders and impulsive fire setters and 127 Finnish psychiatrically interviewed healthy male volunteers were determined. CSF 5-HIAA concentrations were available for 74 alcoholic violent offenders and 25 healthy volunteers. Linkage analysis placed the 5-HT{sub 2C} gene on Xq21, a region that has been previously shown to contain genes for several mental retardation syndromes. The 5-HT{sub 2Ccys}/5-HT{sub 2Cser} genotype frequencies in alcoholic violent offenders and controls differed significantly (0.90/0.10 and 0.82/0.18, respectively, P=0.048). The association was found to be strongest in the violent offenders who did not fulfill the criteria for antisocial personality disorder (5-HT{sub 2Ccys}/5-HT{sub 2Cser} 0.93/0.07, p=0.021). No association was found between CSF 5-HIAA concentrations and 5-HT{sub 2C} genotype. These results implicate a 5-HT{sub 2C} receptor amino acid substitution in predisposition to alcohol abuse and violent behavior in a subgroup of alcoholics.

In the present study behavioral effects of the 5-HT2C serotonin receptor were investigated in different mouse strains. The 5-HT2Creceptoragonist MK-212 applied intraperitoneally induced significant dose-dependent reduction of distance traveled in the open field test in CBA/Lac mice. This effect was receptor-specific because it was inhibited by the 5-HT2Creceptor antagonist RS102221. To study the role of genotype in 5-HT2Creceptor-induced hypolocomotion, locomotor activity of seven inbred mouse strains was measured after MK-212 acute treatment. We found that the 5-HT2Creceptor stimulation by MK-212 decreased distance traveled in the open field test in CBA/Lac, C57Bl/6, C3H/He, and ICR mice, whereas it failed to affect locomotor activity in DBA/2J, Asn, and Balb/c mice. We also compared the interstrain differences in functional response to 5-HT2C and 5-HT2A receptors activation measured by the quantification of receptor-mediated head-twitches. These experiments revealed significant positive correlation between 5-HT2C and 5-HT2A receptors functional responses for all investigated mouse strains. Moreover, we found that 5-HT2A receptor activation with DOI did not change locomotor activity in CBA/Lac mice. Taken together, our data indicate the implication of 5-HT2Creceptors in regulation of locomotor activity and suggest the shared mechanism for functional responses mediated by 5-HT2C and 5-HT2A receptors. PMID:26380122

In the present study behavioral effects of the 5-HT2C serotonin receptor were investigated in different mouse strains. The 5-HT2Creceptoragonist MK-212 applied intraperitoneally induced significant dose-dependent reduction of distance traveled in the open field test in CBA/Lac mice. This effect was receptor-specific because it was inhibited by the 5-HT2Creceptor antagonist RS102221. To study the role of genotype in 5-HT2Creceptor-induced hypolocomotion, locomotor activity of seven inbred mouse strains was measured after MK-212 acute treatment. We found that the 5-HT2Creceptor stimulation by MK-212 decreased distance traveled in the open field test in CBA/Lac, C57Bl/6, C3H/He, and ICR mice, whereas it failed to affect locomotor activity in DBA/2J, Asn, and Balb/c mice. We also compared the interstrain differences in functional response to 5-HT2C and 5-HT2A receptors activation measured by the quantification of receptor-mediated head-twitches. These experiments revealed significant positive correlation between 5-HT2C and 5-HT2A receptors functional responses for all investigated mouse strains. Moreover, we found that 5-HT2A receptor activation with DOI did not change locomotor activity in CBA/Lac mice. Taken together, our data indicate the implication of 5-HT2Creceptors in regulation of locomotor activity and suggest the shared mechanism for functional responses mediated by 5-HT2C and 5-HT2A receptors. PMID:26380122

Although peroxisome proliferator-activated receptoragonists are prescribed to improve cardiovascular risk factors, their cardiovascular safety is controversial. We therefore reviewed the literature to identify landmark randomized controlled trials evaluating the effect of peroxisome proliferator-activated receptor gamma agonists (pioglitazone and rosiglitazone), alpha agonists (fenofibrate and gemfibrozil), and pan agonists (bezafibrate, muraglitazar, ragaglitazar, tesaglitazar, and aleglitazar) on cardiovascular outcomes. Pioglitazone may modestly reduce cardiovascular events but also may increase the risk of bladder cancer. Rosiglitazone increases the risk of myocardial infarction and has been withdrawn in European and restricted in the United States. Fibrates improve cardiovascular outcomes only in select subgroups: fenofibrate in diabetic patients with metabolic syndrome, gemfibrozil in patients with dyslipidemia, and bezafibrate in patients with diabetes or metabolic syndrome. The cardiovascular safety of the new pan agonist aleglitazar, currently in phase II trials, remains to be determined. The heterogenous effects of peroxisome proliferator-activated receptoragonists to date highlight the importance of postmarketing surveillance. The critical question of why peroxisome proliferator-activated receptoragonists seem to improve cardiovascular risk factors without significantly improving cardiovascular outcomes requires further investigation. PMID:22269613

Serotonin 2Creceptor (5-HT2CR) agonists administered systemically attenuate both cocaine-primed and cue-elicited reinstatement of extinguished cocaine-seeking behavior. To further elucidate the function of these receptors in addiction-like processes, this study examined the effects of microinfusing the 5-HT2CR agonist MK212 (0, 10, 30, 100 ng/side/0.2 μl) into the medial prefrontal cortex (mPFC) on cocaine self-administration and reinstatement of extinguished cocaine-seeking behavior. Male Sprague–Dawley rats were trained to self-administer cocaine (0.75 mg/kg, i.v.) paired with light and tone cues. Once responding stabilized, rats received MK212 microinfusions before tests for maintenance of cocaine self-administration. Next, extinction training to reduce cocaine-seeking behavior, defined as responses performed without cocaine reinforcement available, occurred until low extinction baselines were achieved. Rats then received MK212 microinfusions before tests for reinstatement of extinguished cocaine-seeking behavior elicited by cocaine-priming injections (10 mg/kg, i.p.) or response-contingent presentations of the cocaine-associated cues; operant responses during cocaine-primed reinstatement tests produced no consequences. MK212 microinfusions into the prelimbic and infralimbic, but not anterior cingulate, regions of the mPFC dose-dependently attenuated both cocaine-primed and cue-elicited reinstatement of extinguished cocaine-seeking behavior, but did not reliably affect cocaine self-administration. A subsequent experiment showed that the effects of MK212 (100 ng/side/0.2 μl) on reinstatement of extinguished cocaine-seeking behavior were blocked by co-administration of the 5-HT2CR antagonist SB242084 (200 ng/side/0.2 μl). MK212 administered alone into the mPFC as a drug prime produced no discernable effects on cocaine-seeking behavior. These findings suggest that stimulation of 5-HT2CRs in the mPFC attenuates the incentive motivational

Xanomeline [3(3-hexyloxy-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine] has been reported to act as a functionally selective muscarinic partial agonist with potential use in the treatment of Alzheimer's disease. This study examined the functional activity of xanomeline at 5-HT1 and 5-HT2 receptors in native tissue and/or human cloned receptors.Xanomeline had affinity for muscarinic receptors in rat cortical membranes where the ratio of the displacement affinity of [3H]-Quinuclidinyl benzilate vs that of [3H]-Oxotremorine-M was 16, indicative of partial agonist activity. Radioligand binding studies on human cloned receptors confirmed that xanomeline had substantial affinity for M1, M2, M3, M4, M5 receptors and also for 5-HT1 and 5-HT2 receptor subtypes.Carbachol and xanomeline stimulated basal [35S]-GTPγS binding in rat cortical membranes with micromolar affinity. The response to carbachol was attenuated by himbacine and pirenzepine with pA2 of 8.2, 6.9 respectively consistent with the response being mediated, predominantly, via M2 and M4 receptors. Xanomeline-induced stimulation of [35S]-GTPγS binding was inhibited by himbacine with an apparent pKb of 6.3, was not attenuated by pirenzepine up to 3 μM and was inhibited by the selective 5-HT1A antagonist WAY100635 with an apparent pKb of 9.4. These data suggest the agonist effect of xanomeline in this tissue is, in part, via 5-HT1A receptors. Similar studies on human cloned receptors confirmed that xanomeline is an agonist at human cloned 5-HT1A and 5-HT1B receptors.In studies using the fluorescent cytoplasmic Ca2+ indicator FLUO-3AM, xanomeline induced an increase in cytoplasmic Ca2+ concentration in SH-SY5Y cells expressing recombinant human 5-HT2Creceptors. Atropine antagonized this response, consistent with mediation via endogenously-expressed muscarinic receptors. In the presence of atropine, xanomeline antagonized 5-HT-induced cytoplasmic changes in Ca2+ concentration in cells expressing h5

Mechanisms of inverse agonist action at the D2(short) dopamine receptor have been examined. Discrimination of G-protein-coupled and -uncoupled forms of the receptor by inverse agonists was examined in competition ligand-binding studies versus the agonist [3H]NPA at a concentration labelling both G-protein-coupled and -uncoupled receptors. Competition of inverse agonists versus [3H]NPA gave data that were fitted best by a two-binding site model in the absence of GTP but by a one-binding site model in the presence of GTP. K(i) values were derived from the competition data for binding of the inverse agonists to G-protein-uncoupled and -coupled receptors. K(coupled) and K(uncoupled) were statistically different for the set of compounds tested (ANOVA) but the individual values were different in a post hoc test only for (+)-butaclamol. These observations were supported by simulations of these competition experiments according to the extended ternary complex model. Inverse agonist efficacy of the ligands was assessed from their ability to reduce agonist-independent [35S]GTP gamma S binding to varying degrees in concentration-response curves. Inverse agonism by (+)-butaclamol and spiperone occurred at higher potency when GDP was added to assays, whereas the potency of (-)-sulpiride was unaffected. These data show that some inverse agonists ((+)-butaclamol, spiperone) achieve inverse agonism by stabilising the uncoupled form of the receptor at the expense of the coupled form. For other compounds tested, we were unable to define the mechanism. PMID:15735658

Energy and glucose homeostasis are regulated by central serotonin 2Creceptors. These receptors are attractive pharmacological targets for the treatment of obesity; however, the identity of the serotonin 2Creceptor-expressing neurons that mediate the effects of serotonin and serotonin 2Creceptor a...

In this study, we investigated the biochemical mechanisms of agonist action at the G protein-coupled D2 dopamine receptor expressed in Chinese hamster ovary cells. Stimulation of guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding by full and partial agonists was determined at different concentrations of [35S]GTPgammaS (0.1 and 10 nM) and in the presence of different concentrations of GDP. At both concentrations of [35S]GTPgammaS, increasing GDP decreased the [35S]GTPgammaS binding observed with maximally stimulating concentrations of agonist, with partial agonists exhibiting greater sensitivity to the effects of GDP than full agonists. The relative efficacy of partial agonists was greater at the lower GDP concentrations. Concentration-response experiments were performed for a range of agonists at the two [35S]GTPgammaS concentrations and with different concentrations of GDP. At 0.1 nM [35S]GTPgammaS, the potency of both full and partial agonists was dependent on the GDP concentration in the assays. At 10 nM [35S]GTPgammaS, the potency of full agonists exhibited a greater dependence on the GDP concentration, whereas the potency of partial agonists was virtually independent of GDP. We concluded that at the lower [35S]GTPgammaS concentration, the rate-determining step in G protein activation is the binding of [35S]GTPgammaS to the G protein. At the higher [35S]GTPgammaS concentration, for full agonists, [35S]GTPgammaS binding remains the slowest step, whereas for partial agonists, another (GDP-independent) step, probably ternary complex breakdown, becomes rate-determining. PMID:15340043

2,5-Dimethoxy-4-substituted phenylisopropylamines and phenethylamines are 5-hydroxytryptamine (serotonin) (5-HT)(2A/2C) agonists. The former are partial to full agonists, whereas the latter are partial to weak agonists. However, most data come from studies analyzing phospholipase C (PLC)-mediated responses, although additional effectors [e.g., phospholipase A(2) (PLA(2))] are associated with these receptors. We compared two homologous series of phenylisopropylamines and phenethylamines measuring both PLA(2) and PLC responses in Chinese hamster ovary-K1 cells expressing human 5-HT(2A) or 5-HT(2C) receptors. In addition, we assayed both groups of compounds as head shake inducers in rats. At the 5-HT(2C) receptor, most compounds were partial agonists for both pathways. Relative efficacy of some phenylisopropylamines was higher for both responses compared with their phenethylamine counterparts, whereas for others, no differences were found. At the 5-HT(2A) receptor, most compounds behaved as partial agonists, but unlike findings at 5-HT(2C) receptors, all phenylisopropylamines were more efficacious than their phenethylamine counterparts. 2,5-Dimethoxyphenylisopropylamine activated only the PLC pathway at both receptor subtypes, 2,5-dimethoxyphenethylamine was selective for PLC at the 5-HT(2C) receptor, and 2,5-dimethoxy-4-nitrophenethylamine was PLA(2)-specific at the 5-HT(2A) receptor. For both receptors, the rank order of efficacy of compounds differed depending upon which response was measured. The phenylisopropylamines were strong head shake inducers, whereas their phenethylamine congeners were not, in agreement with in vitro results and the involvement of 5-HT(2A) receptors in the head shake response. Our results support the concept of functional selectivity and indicate that subtle changes in ligand structure can result in significant differences in the cellular signaling profile. PMID:17337633

Given its predominant expression in the central nervous system (CNS), 5-hydroxytryptamine (5-HT: serotonin) subtype 6 receptor (5-HT6R) has been considered as a valuable target for the development of CNS drugs with limited side effects. After 2 decades of intense research, numerous selective ligands have been developed to target this receptor; this holds potential interest for the treatment of neuropathological disorders. In fact, some agents (mainly antagonists) are currently undergoing clinical trial. More recently, a series of potent and selective agonists have been developed, and preclinical studies have been conducted that suggest the therapeutic interest of 5-HT6R agonists. This review details the medicinal chemistry of these agonists, highlights their activities, and discusses their potential for treating cognitive issues associated with Alzheimer's disease (AD), depression, or obesity. Surprisingly, some studies have shown that both 5-HT6R agonists and antagonists exert similar procognitive activities. This article summarizes the hypotheses that could explain this paradox. PMID:26099069

beta-Adrenergic receptor (bAR) agonists have been reported to modulate growth in several mammalian and avian species, and bAR agonists presumably exert their physiological action on skeletal muscle cells through this receptor. Because of the importance of bAR regulation on muscle protein metabolism in muscle cells, the objectives of this study were to determine the developmental expression pattern of the bAR population in C2C12 skeletal muscle cells, and to analyze changes in both the quantity and isoform expression of the major muscle protein, myosin. The number of bAR in mononucleated C2C12 cells was approximately 8,000 bAR per cell, which is comparable with the population reported in several other nonmuscle cell types. However, the bar population increased after myoblast fusion to greater than 50,000 bAR per muscle cell equivalent. The reasons for this apparent over-expression of bAR in C2C12 cells is not known. The quantity of myosin also increased after C2C12 myoblast fusion, but the quantity of myosin was less than that reported in primary muscle cell cultures. Finally, at least five different isoforms of myosin heavy chain could be resolved in C2C12 cells, and three of these exhibited either increased or decreased developmental regulation relative to the others. Thus, C2C12 myoblasts undergo developmental regulation of bAR population and myosin heavy chain isoform expression.

Thiazolidinediones (or glitazones) are synthetic PPARgamma (Peroxisome Proliferator-Activated Receptors gamma) ligands with well recognized effects on glucose and lipid metabolism. The clinical use of these PPARgamma agonists in type 2 diabetic patients leads to an improved glycemic control and an inhanced insulin sensitivity, and at least in animal models, to a protective effect on pancreatic beta-cell function. However, they can produce adverse effects, generally mild or moderate, but some of them (mainly peripheral edema and weight gain) may conduct to treatment cessation. Several pharmacological classes are currently in pre-clinical or clinical development, with the objective to retain the beneficial metabolic properties of PPARgamma agonists, either alone or in association with the PPARalpha agonists (fibrates) benefit on lipid profile, but devoid of the side-effects on weight gain and fluid retention. These new pharmacological classes: partial PPARgamma agonists, PPARgamma antagonists, dual PPARalpha/PPARgamma agonists, pan PPARalpha/beta(delta)/gamma agonists, RXR receptoragonists (rexinoids), are presented in this review. Main results from in vitro cell experiments and animal model studies are discussed, as well as the few published short-term studies in type 2 diabetic patients. PMID:15959400

Cys-loop receptors are ligand-gated ion channels that are activated by a structurally diverse array of neurotransmitters, including acetylcholine, serotonin, glycine, and GABA. After the term “chemoreceptor” emerged over 100 years ago, there was some wait until affinity labeling, molecular cloning, functional studies, and X-ray crystallography experiments identified the extracellular interface of adjacent subunits as the principal site of agonist binding. The question of how subtle differences at and around agonist-binding sites of different Cys-loop receptors can accommodate transmitters as chemically diverse as glycine and serotonin has been subject to intense research over the last three decades. This review outlines the functional diversity and current structural understanding of agonist-binding sites, including those of invertebrate Cys-loop receptors. Together, this provides a framework to understand the atomic determinants involved in how these valuable therapeutic targets recognize and bind their ligands. PMID:24795655

We searched for a strong and selective nonprostanoid IP agonist bearing piperidine and benzanilide moieties. Through optimization of substituents on the benzanilide moiety, the crucial part of the agonist, 43 (2-((1-(2-(N-(4-tolyl)benzo[d][1,3]dioxole-5-carboxamido)ethyl)piperidin-4-yl)oxy)acetic acid monohydrate monohydrochloride) was discovered and exhibited strong platelet aggregation inhibition (IC50=21nM) and 100-fold selectivity for IP receptor over other PG receptors. The systemic exposure level and bioavailability after oral administration of 43 were also good in dog. PMID:27133594

A series of novel compounds with two halogen substituents have been designed and synthesized to further optimize the 2-phenylcyclopropylmethylamine scaffold in the quest for drug-like 5-HT2Cagonists. Compound (+)-22a was identified as a potent 5-HT2Creceptoragonist, with good selectivity against the 5-HT2B and the 5-HT2A receptors. ADMET assays showed that compound (+)-22a possessed desirable properties in terms of its microsomal stability, and CYP and hERG inhibition, along with an excellent brain penetration profile. Evaluation of (+)-22a in animal models of schizophrenia-related behaviors revealed that it had a desirable activity profile, as it reduced d-amphetamine-stimulated hyperlocomotion in the open field test, it restored d-amphetamine-disrupted prepulse inhibition, it induced cognitive improvements in the novel object recognition memory test in NR1-KD animals, and it produced very little catalepsy relative to haloperidol. These data support the further development of (+)-22a as a drug candidate for the treatment of schizophrenia. PMID:26704965

Lorcaserin (APD356) is a potent, selective 5-HT(2C) agonist with ~15-fold and 100-fold selectivity vs. 5-HT(2A) and 5-HT(2B) receptors, respectively. This study evaluated the safety and efficacy of lorcaserin for weight reduction in obese patients during a 12-week period. The randomized, double-blind, placebo-controlled, parallel-arm study enrolled 469 men and women between ages 18 and 65 and with BMI 30-45 kg/m(2). Patients received placebo, lorcaserin 10 mg q.d., lorcaserin 15 mg q.d., or lorcaserin 10 mg b.i.d. for 12 weeks, and were counseled to maintain their usual diet and activity. The primary end point was change in weight from baseline to day 85 by completer analysis. Safety analyses included echocardiograms at Screening and day 85/study exit. Lorcaserin was associated with progressive weight loss of 1.8 kg, 2.6 kg, and 3.6 kg at 10 mg q.d., 15 mg q.d., and 10 mg b.i.d., respectively, compared to placebo weight loss of 0.3 kg (P < 0.001 for each group). Similar results were seen by intent-to-treat last observation-carried forward (ITT-LOCF) analysis. The proportions of completers achieving > or =5% of initial body weight were 12.8, 19.5, 31.2, and 2.3% in the 10 mg q.d., 15 mg q.d., 10 mg b.i.d., and placebo groups, respectively. The most frequent adverse events (AEs) were transient headache, nausea, and dizziness. Echocardiograms showed no apparent drug-related effects on heart valves or pulmonary artery pressure (PAP). Lorcaserin was well tolerated and efficacious for weight reduction in this 12-week study. Longer-term trials employing behavior modification will be needed to more fully assess its safety and efficacy. PMID:19057523

Inasmuch as the neurohormone melatonin is synthetically derived from serotonin (5-HT), a close interrelationship between both has long been suspected. The present study reveals a hitherto unrecognized cross-talk mediated via physical association of melatonin MT2 and 5-HT2Creceptors into functional heteromers. This is of particular interest in light of the "synergistic" melatonin agonist/5-HT2C antagonist profile of the novel antidepressant agomelatine. A suite of co-immunoprecipitation, bioluminescence resonance energy transfer, and pharmacological techniques was exploited to demonstrate formation of functional MT2 and 5-HT2Creceptor heteromers both in transfected cells and in human cortex and hippocampus. MT2/5-HT2C heteromers amplified the 5-HT-mediated Gq/phospholipase C response and triggered melatonin-induced unidirectional transactivation of the 5-HT2C protomer of MT2/5-HT2C heteromers. Pharmacological studies revealed distinct functional properties for agomelatine, which shows "biased signaling." These observations demonstrate the existence of functionally unique MT2/5-HT2C heteromers and suggest that the antidepressant agomelatine has a distinctive profile at these sites potentially involved in its therapeutic effects on major depression and generalized anxiety disorder. Finally, MT2/5-HT2C heteromers provide a new strategy for the discovery of novel agents for the treatment of psychiatric disorders. PMID:25770211

Medicines that activate cannabinoid CB(1) and CB(2) receptor are already in the clinic. These are Cesamet (nabilone), Marinol (dronabinol; Delta(9)-tetrahydrocannabinol) and Sativex (Delta(9)-tetrahydrocannabinol with cannabidiol). The first two of these medicines can be prescribed to reduce chemotherapy-induced nausea and vomiting. Marinol can also be prescribed to stimulate appetite, while Sativex is prescribed for the symptomatic relief of neuropathic pain in adults with multiple sclerosis and as an adjunctive analgesic treatment for adult patients with advanced cancer. One challenge now is to identify additional therapeutic targets for cannabinoid receptoragonists, and a number of potential clinical applications for such agonists are mentioned in this review. A second challenge is to develop strategies that will improve the efficacy and/or the benefit-to-risk ratio of a cannabinoid receptoragonist. This review focuses on five strategies that have the potential to meet either or both of these objectives. These are strategies that involve: (i) targeting cannabinoid receptors located outside the blood-brain barrier; (ii) targeting cannabinoid receptors expressed by a particular tissue; (iii) targeting up-regulated cannabinoid receptors; (iv) targeting cannabinoid CB(2) receptors; or (v) 'multi-targeting'. Preclinical data that justify additional research directed at evaluating the clinical importance of each of these strategies are also discussed. PMID:19226257

Medicines that activate cannabinoid CB1 and CB2 receptor are already in the clinic. These are Cesamet® (nabilone), Marinol® (dronabinol; Δ9-tetrahydrocannabinol) and Sativex® (Δ9-tetrahydrocannabinol with cannabidiol). The first two of these medicines can be prescribed to reduce chemotherapy-induced nausea and vomiting. Marinol® can also be prescribed to stimulate appetite, while Sativex® is prescribed for the symptomatic relief of neuropathic pain in adults with multiple sclerosis and as an adjunctive analgesic treatment for adult patients with advanced cancer. One challenge now is to identify additional therapeutic targets for cannabinoid receptoragonists, and a number of potential clinical applications for such agonists are mentioned in this review. A second challenge is to develop strategies that will improve the efficacy and/or the benefit-to-risk ratio of a cannabinoid receptoragonist. This review focuses on five strategies that have the potential to meet either or both of these objectives. These are strategies that involve: (i) targeting cannabinoid receptors located outside the blood-brain barrier; (ii) targeting cannabinoid receptors expressed by a particular tissue; (iii) targeting up-regulated cannabinoid receptors; (iv) targeting cannabinoid CB2 receptors; or (v) ‘multi-targeting’. Preclinical data that justify additional research directed at evaluating the clinical importance of each of these strategies are also discussed. PMID:19226257

Methamphetamine profoundly increases brain monoamines and is a widely abused psychostimulant. The effects of methamphetamine self-administration on neuron function are not known for the nucleus accumbens, a brain region involved in addictive behaviors, including drug-seeking. One therapeutic target showing preclinical promise at attenuating psychostimulant-seeking is 5-HT2Creceptors; however, the effects of 5-HT2Creceptor ligands on neuronal physiology are unclear. 5-HT2Creceptor agonism decreases psychostimulant-mediated behaviors, and the putative 5-HT2Creceptor inverse agonist, SB 206553, attenuates methamphetamine-seeking in rats. To ascertain the effects of methamphetamine, and 5-HT2Creceptor inverse agonism and agonism, on neuronal function in the nucleus accumbens, we evaluated methamphetamine, SB 206553, and the 5-HT2Creceptoragonist and Ro 60-0175, on neuronal excitability within the accumbens shell subregion using whole-cell current-clamp recordings in forebrain slices ex vivo. We reveal that methamphetamine self-administration decreased generation of evoked action potentials. In contrast, SB 206553 and Ro 60-0175 increased evoked spiking, effects that were prevented by the 5-HT2Creceptor antagonist, SB 242084. We also assessed signaling mechanisms engaged by 5-HT2Creceptors, and determined that accumbal 5-HT2Creceptors stimulated Gq, but not Gi/o. These findings demonstrate that methamphetamine-induced decreases in excitability of neurons within the nucleus accumbens shell were abrogated by both 5-HT2C inverse agonism and agonism, and this effect likely involved activation of Gq–mediated signaling pathways. PMID:25229719

Objectives Glucagon-like peptide-1 (GLP-1) receptoragonists have been used to treat type 2 diabetes for almost a decade, and new treatments in this class have recently been introduced. The purpose of this study was to examine perceptions of GLP-1 receptoragonists among physicians who treat patients with type 2 diabetes in the UK. Methods A total of 670 physicians (226 diabetes specialists; 444 general practice [GP] physicians) completed a survey in 2014. Results Almost all physicians had prescribed GLP-1 receptoragonists (95.4% total sample; 99.1% specialists; 93.5% GP), most frequently to patients whose glucose levels are not adequately controlled with oral medications (85.9% of physicians) and obese/overweight patients (83.7%). Physicians' most common reasons for prescribing a GLP-1 receptoragonist were: associated with weight loss (65.8%), good efficacy (55.7%), less hypoglycemia risk than insulin (55.2%), not associated with weight gain (34.5%), and better efficacy than oral medications (32.7%). Factors that most commonly cause hesitation when prescribing this class were: not considered first line therapy according to guidelines (56.9%), injectable administration (44.6%), cost (36.7%), gastrointestinal side effects (33.4%), and risk of pancreatitis (26.7%). Almost all specialists (99.1%) believed they had sufficient knowledge to prescribe a GLP-1 receptoragonist, compared with 76.1% of GPs. Conclusions Results highlight the widespread use of GLP-1 receptoragonists for treatment of type 2 diabetes in the UK. However, almost a quarter of GPs reported that they do not have enough knowledge to prescribe GLP-1s, suggesting a need for increased dissemination of information to targeted groups of physicians. Study limitations were that the generalizability of the clinician sample is unknown; survey questions required clinicians to select answers from multiple response options rather than generating the responses themselves; and responses to this survey conducted

The existence of a third tachykinin receptor (SP-N) in the mammalian nervous system was demonstrated by development of highly selective agonists. Systematic N-methylation of individual peptide bonds in the C-terminal hexapeptide of substance P gave rise to agonists which specifically act on different receptor subtypes. The most selective analog of this series, succinyl-[Asp6,Me-Phe8]SP6-11, elicits half-maximal contraction of the guinea pig ileum through the neuronal SP-N receptor at a concentration of 0.5 nM. At least 60,000-fold higher concentrations of this peptide are required to stimulate the other two tachykinin receptors (SP-P and SP-E). The action of selective SP-N agonists in the guinea pig ileum is antagonized by opioid peptides, suggesting a functional counteraction between opiate and SP-N receptors. These results indicate that the tachykinin receptors are distinct entities which may mediate different physiological functions. PMID:2431898

Hypersecretion of norepinephrine (NE) and angiotensin II (AngII) is a hallmark of major prevalent cardiovascular diseases that contribute to cardiac pathophysiology and morbidity. Herein, we explore whether heterodimerization of presynaptic AngII AT1 receptor (AT1-R) and NE α2C-adrenergic receptor (α2C-AR) could underlie their functional cross-talk to control NE secretion. Multiple bioluminescence resonance energy transfer and protein complementation assays allowed us to accurately probe the structures and functions of the α2C-AR–AT1-R dimer promoted by ligand binding to individual protomers. We found that dual agonist occupancy resulted in a conformation of the heterodimer different from that induced by active individual protomers and triggered atypical Gs-cAMP–PKA signaling. This specific pharmacological signaling unit was identified in vivo to promote not only NE hypersecretion in sympathetic neurons but also sympathetic hyperactivity in mice. Thus, we uncovered a new process by which GPCR heterodimerization creates an original functional pharmacological entity and that could constitute a promising new target in cardiovascular therapeutics. PMID:25706338

Absence seizures (ASs) are the hallmark of childhood/juvenile absence epilepsy. Monotherapy with first-line anti-absence drugs only controls ASs in 50% of patients, indicating the need for novel therapeutic targets. Since serotonin family-2 receptors (5-HT2Rs) are known to modulate neuronal activity in the cortico-thalamo-cortical loop, the main network involved in AS generation, we investigated the effect of selective 5-HT2AR and 5-HT2CR ligands on ASs in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS), a well established polygenic rat model of these non-convulsive seizures. GAERS rats were implanted with fronto-parietal EEG electrodes under general anesthesia, and their ASs were later recorded under freely moving conditions before and after intraperitoneal administration of various 5-HT2AR and 5-HT2CR ligands. The 5-HT2A agonist TCB-2 dose-dependently decreased the total time spent in ASs, an effect that was blocked by the selective 5-HT2A antagonist MDL11,939. Both MDL11,939 and another selective 5-HT2A antagonist (M100,907) increased the length of individual seizures when injected alone. The 5-HT2Cagonists lorcaserin and CP-809,101 dose-dependently suppressed ASs, an effect blocked by the selective 5-HT2C antagonist SB 242984. In summary, 5-HT2ARs and 5-HT2CRs negatively control the expression of experimental ASs, indicating that selective agonists at these 5-HT2R subtypes might be potential novel anti-absence drugs. PMID:27085605

Fleas are significant ectoparasites of small animals. They can be a severe irritant to animals and serve as a vector for a number of infectious diseases. In this article, we discuss the pharmacological characteristics of four insect nicotinic acetylcholine receptor (nAChR) agonists used as fleacides in dogs and cats, which include three neonicotinoids (imidacloprid, nitenpyram, and dinotefuran) and spinosad. Insect nAChR agonists are one of the most important new classes of insecticides, which are used to control sucking insects both on plants and on companion animals. These new compounds provide a new approach for practitioners to safely and effectively eliminate fleas. PMID:20646191

G protein-coupled receptors constitute the largest family of membrane receptors and modulate almost every physiological process in humans. Binding of agonists to G protein-coupled receptors induces a shift from inactive to active receptor conformations. Biophysical studies of the dynamic equilibrium of receptors suggest that a portion of receptors can remain in inactive states even in the presence of saturating concentrations of agonist and G protein mimetic. However, the molecular details of agonist-bound inactive receptors are poorly understood. Here we use the model of bitopic orthosteric/allosteric (i.e. dualsteric) agonists for muscarinic M2 receptors to demonstrate the existence and function of such inactive agonist·receptor complexes on a molecular level. Using all-atom molecular dynamics simulations, dynophores (i.e. a combination of static three-dimensional pharmacophores and molecular dynamics-based conformational sampling), ligand design, and receptor mutagenesis, we show that inactive agonist·receptor complexes can result from agonist binding to the allosteric vestibule alone, whereas the dualsteric binding mode produces active receptors. Each agonist forms a distinct ligand binding ensemble, and different agonist efficacies depend on the fraction of purely allosteric (i.e. inactive) versus dualsteric (i.e. active) binding modes. We propose that this concept may explain why agonist·receptor complexes can be inactive and that adopting multiple binding modes may be generalized also to small agonists where binding modes will be only subtly different and confined to only one binding site. PMID:27298318

Saralasin and sarile, extensively studied over the past 40 years as angiotensin II (Ang II) receptor blockers, induce neurite outgrowth in a NG108-15 cell assay to a similar extent as the endogenous Ang II. In their undifferentiated state, these cells express mainly the AT2 receptor. The neurite outgrowth was inhibited by preincubation with the AT2 receptor selective antagonist PD 123,319, which suggests that the observed outgrowth was mediated by the AT2 receptor. Neither saralasin nor sarile reduced the neurite outgrowth induced by Ang II proving that the two octapeptides do not act as antagonists at the AT2 receptor and may be considered as AT2 receptoragonists. PMID:25313325

Nasal congestion is one of the most troublesome symptoms of many upper airways diseases. We characterized the effect of selective α2c-adrenergic agonists in animal models of nasal congestion. In porcine mucosa tissue, compound A and compound B contracted nasal veins with only modest effects on arteries. In in vivo experiments, we examined the nasal decongestant dose-response characteristics, pharmacokinetic/pharmacodynamic relationship, duration of action, potential development of tolerance, and topical efficacy of α2c-adrenergic agonists. Acoustic rhinometry was used to determine nasal cavity dimensions following intranasal compound 48/80 (1%, 75 µl). In feline experiments, compound 48/80 decreased nasal cavity volume and minimum cross-sectional areas by 77% and 40%, respectively. Oral administration of compound A (0.1-3.0 mg/kg), compound B (0.3-5.0 mg/kg), and d-pseudoephedrine (0.3 and 1.0 mg/kg) produced dose-dependent decongestion. Unlike d-pseudoephedrine, compounds A and B did not alter systolic blood pressure. The plasma exposure of compound A to produce a robust decongestion (EC(80)) was 500 nM, which related well to the duration of action of approximately 4.0 hours. No tolerance to the decongestant effect of compound A (1.0 mg/kg p.o.) was observed. To study the topical efficacies of compounds A and B, the drugs were given topically 30 minutes after compound 48/80 (a therapeutic paradigm) where both agents reversed nasal congestion. Finally, nasal-decongestive activity was confirmed in the dog. We demonstrate that α2c-adrenergic agonists behave as nasal decongestants without cardiovascular actions in animal models of upper airway congestion. PMID:24492651

The 5-HT2Creceptor (R) displays a widespread distribution in the CNS and is involved in the action of 5-HT in all brain areas. Knowledge of its functional role in the CNS pathophysiology has been impaired for many years due to the lack of drugs capable of discriminating among 5-HT2R subtypes, and to a lesser extent to the 5-HT1B, 5-HT5, 5-HT6 and 5-HT7Rs. The situation has changed since the mid-90s due to the increased availability of new and selective synthesized compounds, the creation of 5-HT2C knock out mice, and the progress made in molecular biology. Many pharmacological classes of drugs including antipsychotics, antidepressants and anxiolytics display affinities toward 5-HT2CRs and new 5-HT2C ligands have been developed for various neuropsychiatric disorders. The 5-HT2CR is presumed to mediate tonic/constitutive and phasic controls on the activity of different central neurobiological networks. Preclinical data illustrate this complexity to a point that pharmaceutical companies developed either agonists or antagonists for the same disease. In order to better comprehend this complexity, this review will briefly describe the molecular pharmacology of 5-HT2CRs, as well as their cellular impacts in general, before addressing its central distribution in the mammalian brain. Thereafter, we review the preclinical efficacy of 5-HT2C ligands in numerous behavioral tests modeling human diseases, highlighting the multiple and competing actions of the 5-HT2CRs in neurobiological networks and monoaminergic systems. Notably, we will focus this evidence in the context of the physiopathology of psychiatric and neurological disorders including Parkinson's disease, levodopa-induced dyskinesia, and epilepsy. PMID:26617215

We have screened a variety of L-amino acid peptide libraries against the extracellular domain of the human thrombopoietin (HuTPO) receptor, c-Mpl. A large number of peptide ligands were recovered and categorized into two families. Peptides from each family compete with the binding of HuTPO and with the binding of peptides from the other familiy. Representative peptides were synthesized and found to activate the full-length HuTPO receptor expressed in Ba/F3 cells to promote proliferation. These peptide families show no apparent homology to the primary sequence of TPO. We have focused our optimization efforts on one of the peptides, a linear 14-mer (IEGPTLRQWLAARA) with an IC50 of 2 nM in a competition binding assay and an EC50 of 400 nM in the proliferation assay. In order to enhance the potency of the compound, we constructed dimeric peptides by linking the carboxy-termini of the 14-mers to a lysine branch. These molecules exhibited slightly higher affinity (0.5 nM) and greatly increased potency (0.1 nM). The EC50 of the dimeric peptide was equivalent to that of the 332 aa form of baculovirus-expressed recombinant HuTPO. As previously shown for the erythropoietin-mimetic peptides, the TPO-mimetic peptides probably activate the TPO receptor by binding and inducing receptor dimerization. This supposition is supported by the observation that covalent dimerization of the peptide enhances its potency by 4,000-fold over that of the monomer. The peptide dimer is also active in stimulating in vitro proliferation of progenitors and maturation of megakaryocytes from human bone marrow, and in promoting an increase in platelet count when administered to normal mice. PMID:11012174

Structural studies on G protein-coupled receptors (GPCRs) provide important insights into the architecture and function of these important drug targets. However, the crystallization of GPCRs in active states is particularly challenging, requiring the formation of stable and conformationally homogeneous ligand-receptor complexes. Native hormones, neurotransmitters, and synthetic agonists that bind with low affinity are ineffective at stabilizing an active state for crystallogenesis. To promote structural studies on the pharmacologically highly relevant class of aminergic GPCRs, we here present the development of covalently binding molecular tools activating Gs-, Gi-, and Gq-coupled receptors. The covalent agonists are derived from the monoamine neurotransmitters noradrenaline, dopamine, serotonin, and histamine, and they were accessed using a general and versatile synthetic strategy. We demonstrate that the tool compounds presented herein display an efficient covalent binding mode and that the respective covalent ligand-receptor complexes activate G proteins comparable to the natural neurotransmitters. A crystal structure of the β2-adrenoreceptor in complex with a covalent noradrenaline analog and a conformationally selective antibody (nanobody) verified that these agonists can be used to facilitate crystallogenesis. PMID:25006259

The interaction of a small molecule made in one cell with a large receptor made in another is the signature event of cell signaling. Understanding the structure and energy changes associated with agonist activation is important for engineering drugs, receptors and synapses. The nicotinic acetylcholine receptor (AChR) is a ∼300kD ion channel that binds the neurotransmitter acetylcholine (ACh) and other cholinergic agonists to elicit electrical responses in the central and peripheral nervous systems. This mini-review is in two sections. First, general concepts of skeletal muscle AChR operation are discussed in terms of energy landscapes for conformational change. Second, adult vs. fetal AChRs are compared with regard to interaction energies between ACh and agonist-site side chains, measured by single-channel electrophysiology and molecular dynamics simulations. The five aromatic residues that form the core of each agonist binding site can be divided into two working groups, a triad (led by αY190) that behaves similarly at all sites and a coupled pair (led by γW55) that has a large influence on affinity only in fetal AChRs. Each endplate AChR has 5 homologous subunits, two of α(1) and one each of β, δ, and either γ (fetal) or ϵ (adult). These nicotinic AChRs have only 2 functional agonist binding sites located in the extracellular domain, at αδ and either αγ or αϵ subunit interfaces. The receptor undergoes a reversible, global isomerization between structures called C and O. The C shape does not conduct ions and has a relatively low affinity for ACh, whereas O conducts cations and has a higher affinity. When both agonist sites are empty (filled only with water) the probability of taking on the O conformation (PO) is low, <10(-6). When ACh molecules occupy the agonist sites the C→O opening rate constant and C↔O gating equilibrium constant increase dramatically. Following a pulse of ACh at the nerve-muscle synapse, the endplate current rises rapidly

Molecular probe tool compounds for the Sphingosine 1-phosphate receptor 2 (S1PR2) are important for investigating the multiple biological processes in which the S1PR2 receptor has been implicated. Amongst these are NF-κB-mediated tumor cell survival and fibroblast chemotaxis to fibronectin. Here we report our efforts to identify selective chemical probes for S1PR2 and their characterization. We employed high throughput screening to identify two compounds which activate the S1PR2 receptor. SAR optimization led to compounds with high nanomolar potency. These compounds, XAX-162 and CYM-5520, are highly selective and do not activate other S1P receptors. Binding of CYM-5520 is not competitive with the antagonist JTE-013. Mutation of receptor residues responsible for binding to the zwitterionic headgroup of sphingosine 1-phosphate (S1P) abolishes S1P activation of the receptor, but not activation by CYM-5520. Competitive binding experiments with radiolabeled S1P demonstrate that CYM-5520 is an allosteric agonist and does not displace the native ligand. Computational modeling suggests that CYM-5520 binds lower in the orthosteric binding pocket, and that co-binding with S1P is energetically well tolerated. In summary, we have identified an allosteric S1PR2 selective agonist compound. PMID:23849205

The previously reported pyrrolidine class of progesterone receptor partial agonists demonstrated excellent potency but suffered from serious liabilities including hERG blockade and high volume of distribution in the rat. The basic pyrrolidine amine was intentionally converted to a sulfonamide, carbamate, or amide to address these liabilities. The evaluation of the degree of partial agonism for these non-basic pyrrolidine derivatives and demonstration of their efficacy in an in vivo model of endometriosis is disclosed herein.

The bioactive metabolite sphingosine-1-phosphate (S1P), a product of sphingosine kinases (SphKs), mediates diverse biological processes such as cell differentiation, proliferation, survival and angiogenesis. A fluorinated analogue of S1P receptoragonist has been synthesized by utilizing a ring opening reaction of oxacycles by a lithiated difluoromethylphosphonate anion as the key reaction. In vitro activity of this S1P analogue is also reported. PMID:25047939

Dopamine-deficient (DA-/-) mice were created by targeted inactivation of the tyrosine hydroxylase gene in dopaminergic neurons. The locomotor activity response of these mutants to dopamine D1 or D2 receptoragonists and l-3,4-dihydroxyphenylalanine (l-DOPA) was 3- to 13-fold greater than the response elicited from wild-type mice. The enhanced sensitivity of DA-/- mice to agonists was independent of changes in steady-state levels of dopamine receptors and the presynaptic dopamine transporter as measured by ligand binding. The acute behavioral response of DA-/- mice to a dopamine D1 receptoragonist was correlated with c-fos induction in the striatum, a brain nucleus that receives dense dopaminergic input. Chronic replacement of dopamine to DA-/- mice by repeated l-DOPA administration over 4 d relieved the hypersensitivity of DA-/- mutants in terms of induction of both locomotion and striatal c-fos expression. The results suggest that the chronic presence of dopaminergic neurotransmission is required to dampen the intracellular signaling response of striatal neurons. PMID:10844009

The serotonin (5-hydroxytryptamine, 5-HT) 5-HT2 G protein-coupled receptor (GPCR) family consists of types 2A, 2B, and 2C that share ∼75% transmembrane (TM) sequence identity. Agonists for 5-HT2Creceptors are under development for psychoses; whereas, at 5-HT2A receptors, antipsychotic effects are associated with antagonists - in fact, 5-HT2A agonists can cause hallucinations and 5-HT2B agonists cause cardiotoxicity. It is known that 5-HT2A TM6 residues W6.48, F6.51, and F6.52 impact ligand binding and function; however, ligand interactions with these residues at the 5-HT2Creceptor have not been reported. To predict and validate molecular determinants for 5-HT2C-specific activation, results from receptor homology modelling, ligand docking, and molecular dynamics simulation studies were compared with experimental results for ligand binding and function at wild type and W6.48A, F6.51A, and F6.52A point-mutated 5-HT2Creceptors.

The cytochrome P450, CYP2C8, metabolizes more than 60 clinically used drugs as well as endogenous substances including retinoic acid and arachidonic acid. However, predictive factors for interindividual variability in the efficacy and toxicity of CYP2C8 drug substrates are essentially lacking. Recently we demonstrated that peroxisome proliferator-activated receptor alpha (PPARα), a nuclear receptor primarily involved in control of lipid and energy homeostasis directly regulates the transcription of CYP3A4. Here we investigated the potential regulation of CYP2C8 by PPARα. Two linked intronic SNPs in PPARα (rs4253728, rs4823613) previously associated with hepatic CYP3A4 status showed significant association with CYP2C8 protein level in human liver samples (N = 150). Furthermore, siRNA-mediated knock-down of PPARα in HepaRG human hepatocyte cells resulted in up to ∼60 and ∼50% downregulation of CYP2C8 mRNA and activity, while treatment with the PPARα agonist WY14,643 lead to an induction by >150 and >100%, respectively. Using chromatin immunoprecipitation scanning assay we identified a specific upstream gene region that is occupied in vivo by PPARα. Electromobility shift assay demonstrated direct binding of PPARα to a DR-1 motif located at positions –2762/–2775 bp upstream of the CYP2C8 transcription start site. We further validated the functional activity of this element using luciferase reporter gene assays in HuH7 cells. Moreover, based on our previous studies we demonstrated that WNT/β-catenin acts as a functional inhibitor of PPARα-mediated inducibility of CYP2C8 expression. In conclusion, our data suggest direct involvement of PPARα in both constitutive and inducible regulation of CYP2C8 expression in human liver, which is further modulated by WNT/β-catenin pathway. PPARA gene polymorphism could have a modest influence on CYP2C8 phenotype. PMID:26582990

Cell-based bioassays can be used to predict the eventual biological activity of a substance on a living organism. In vitro reporter gene bioassays are based on recombinant vertebrate cell lines or yeast strains and especially the latter are easy-to-handle, cheap, and fast. Moreover, yeast cells do not express estrogen, androgen, progesterone or glucocorticoid receptors, and are thus powerful tools in the development of specific reporter gene systems that are devoid of crosstalk from other hormone pathways. This chapter describes our experience with an in-house developed RIKILT yeast estrogen bioassay for testing estrogen receptoragonists and antagonists, focusing on the applicability of the latter. PMID:26585147

Cannabinoid receptoragonists, such as Δ9-tetrahydrocannabinol (Δ9-THC), enhance the antinociceptive effects of μ-opioid receptoragonists, which suggests that combining cannabinoids with opioids would improve pain treatment. Combinations with lower efficacy agonists might be preferred and could avoid adverse effects associated with large doses; however, it is unclear whether interactions between opioids and cannabinoids vary across drugs with different efficacy. The antinociceptive effects of μ-opioid receptoragonists alone and in combination with cannabinoid receptoragonists were studied in rhesus monkeys (n = 4) using a warm water tail withdrawal procedure. Etorphine, fentanyl, morphine, buprenorphine, nalbuphine, Δ9-THC, and CP 55,940 (2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl) cyclohexyl]-5-(2-methyloctan-2-yl)phenol) each increased tail withdrawal latency. Pretreatment with doses of Δ9-THC (1.0 mg/kg) or CP 55,940 (0.032 mg/kg) that were ineffective alone shifted the fentanyl dose-effect curve leftward 20.6- and 52.9-fold, respectively, and the etorphine dose-effect curve leftward 12.4- and 19.6-fold, respectively. Δ9-THC and CP 55,940 shifted the morphine dose-effect curve leftward only 3.4- and 7.9-fold, respectively, and the buprenorphine curve only 5.4- and 4.1-fold, respectively. Neither Δ9-THC nor CP 55,940 significantly altered the effects of nalbuphine. Cannabinoid receptoragonists increase the antinociceptive potency of higher efficacy opioid receptoragonists more than lower efficacy agonists; however, because much smaller doses of each drug can be administered in combinations while achieving adequate pain relief and that other (e.g., abuse-related) effects of opioids do not appear to be enhanced by cannabinoids, these results provide additional support for combining opioids with cannabinoids to treat pain. PMID:25194020

Cryptochinones A-D are tetrahydroflavanones isolated from the leaves of Cryptocarya chinensis, an evergreen tree whose extracts are believed to have a variety of health benefits. The origin of their possible bioactivity is unclear. The farnesoid X receptor (FXR) is a member of nuclear receptor superfamily that has been widely targeted for developing treatments for chronic liver disease and for hyperglycemia. We studied whether cryptochinones A-D, which are structurally similar to known FXR ligands, may act at this target. Indeed, in mammalian one-hybrid and transient transfection reporter assays, cryptochinones A-D transactivated FXR to modulate promoter action including GAL4, SHP, CYP7A1, and PLTP promoters in dose-dependent manner, while they exhibited similar agonistic activity as chenodeoxycholic acid (CDCA), an endogenous FXR agonist. Through molecular modeling docking studies we evaluated their ability to bind to the FXR ligand binding pocket. Our results indicate that cryptochinones A-D can behave as FXR agonists. PMID:25127166

SAR was used to further develop an indazole class of non-steroidal glucocorticoid receptoragonists aided by a GR LBD (ligand-binding domain)-agonist co-crystal structure described in the accompanying paper. Progress towards discovering a dissociated GR agonist guided by human in vitro assays biased the optimization of this compound series towards partial agonists that possessed excellent selectivity against other nuclear hormone receptors. PMID:23916594

CP-533,536, (3-{[(4-tert-butyl-benzyl)-(pyridine-3-sulfonyl)-amino]-methyl}-phenoxy)-acetic acid (1), an EP2 receptor-selective prostaglandin E2 agonist, is being developed to aid in the healing of bone fractures. To support the development of this program, in vitro metabolism of 1 was investigated in human liver microsomes and major recombinant human cytochrome P450 (P450) isoforms. 1 was metabolized in vitro by at least three recombinant human P450s: CYP3A4, CYP3A5, and CYP2C8. The turnover of 1 was NADPH-dependent and was completely inhibited by ketoconazole and quercetin in the CYP3A4/5 and CYP2C8 incubations, respectively. The major metabolic pathways were caused by oxidation of the tert-butyl moiety to form the omega-hydroxy metabolite (M4), oxidation of the pyridine moiety, and/or N-dealkylation of the methylphenoxy acetic acid moiety. The alcohol metabolite M4 was further oxidized to the corresponding carboxylic acid M3. In addition to these pathways, three unusual metabolites (M22, M23, and M26) resulting from C-demethylation of the tert-butyl group were identified using high-resolution liquid chromatography/tandem mass spectrometry and liquid chromatography/mass spectrometry/NMR. The C-demethylated metabolites were not detected on incubation of carboxylic acid metabolite M3 with either human liver microsomes or CYP3A/2C8 isoforms, suggesting that these metabolites were not derived from decarboxylation of M3. A possible mechanism for C-demethylation may involve the oxidation of M4 to form an aldehyde metabolite (M24), followed by P450-mediated deformylation, to give an unstable carbon-centered radical and formic acid. The carbon-centered radical intermediate then undergoes either oxygen rebound to form an alcohol metabolite M23 or hydrogen abstraction leading to an olefin metabolite M26. PMID:18653741

The interaction of a radiolabeled muscarinic cholinergic receptoragonist, (methyl-/sup 3/H)oxotremorine acetate ((/sup 3/H)OXO), with a washed membrane preparation derived from rat heart, has been studied. In binding assays at 4 degrees C, the rate constants for association and dissociation of (/sup 3/H)OXO were 2 X 10(7) M-1 min-1 and 5 X 10(-3) min-1, respectively, Saturation binding isotherms indicated that binding was to a single population of sites with a Kd of approximately 300 pM. The density of (/sup 3/H)OXO binding sites (90-100 fmol/mg of protein) was approximately 75% of that determined for the radiolabeled receptor antagonist (/sup 3/H)quinuclidinyl benzilate. Both muscarinic receptoragonists and antagonists inhibited the binding of (/sup 3/H)OXO with high affinity and Hill slopes of approximately one. Guanine nucleotides completely inhibited the binding of (/sup 3/H)OXO. This effect was on the maximum binding (Bmax) of (/sup 3/H)OXO with no change occurring in the Kd; the order of potency for five nucleotides was guanosine 5'-O-(3-thio-triphosphate) greater than 5'-guanylylimidodiphosphate greater than GTP greater than or equal to guanosine/diphosphate greater than GMP. The (/sup 3/H)OXO-induced interaction of muscarinic receptors with a guanine nucleotide binding protein was stable to solubilization. That is, membrane receptors that were prelabeled with (/sup 3/H)OXO could be solubilized with digitonin, and the addition of guanine nucleotides to the soluble, (/sup 3/H)OXO-labeled complex resulted in dissociation of (/sup 3/H)OXO from the receptor. Pretreatment of membranes with relatively low concentrations of N-ethylmaleimide inhibited (/sup 3/H)OXO binding by 85% with no change in the Kd of (/sup 3/H)OXO, and with no effect on (/sup 3/H)quinuclidinyl benzilate binding.

The present experiments were performed to investigate the effects of dopamine D1 receptoragonists given alone or in combination with dopamine D2 receptoragonists on body temperature in rats. The selective dopamine D1 receptoragonist, 1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol (SK&F38393), produced hyperthermia. However, the dopamine D2 receptoragonist, B-HT 920 (talipexole), and the newly synthesized dopamine D2 receptoragonist, (S)-2-amino-4,5,6,7-tetrahydro-6-propylamino-benzothiazole (SND 919), did not change the temperature. Interestingly, the SK&F38393-induced hyperthermia was enhanced by talipexole and SND 919. The drastic hyperthermia induced by combined administration of dopamine D1 and D2 receptoragonists was blocked by either the dopamine D1 receptor antagonist, SCH23390, or the dopamine D2 receptor antagonist, spiperone. On the other hand, treatment with prazosin, yohimbine, propranolol, scopolamine, or methysergide failed to affect the marked hyperthermia. The present results suggest that a functional link between dopamine D1 and D2 receptors may be synergistic in the regulation of body temperature and that concurrent stimulation of both dopamine D1 and D2 receptors thereby produces marked hyperthermia in the rat. PMID:1361996

Pyrabactin resistance (PYR) 1 and its relatives belong to a family of soluble abscisic acid (ABA) receptors that inhibit type 2C protein phosphatases (PP2C) when in their agonist-stabilized conformation. Given their switch-like properties, we envisioned that mutations that stabilize their agonist-bound conformation could be used to activate signaling in vivo. To identify such mutations, we subjected PYR1 to site-saturation mutagenesis at 39 highly conserved residues that participate in ABA or PP2C contacts. All 741 possible single amino acid substitutions at these sites were tested to identify variants that increase basal PYR1-PP2C interactions, which uncovered activating mutations in 10 residues that preferentially cluster in PYR1's gate loop and C-terminal helix. The mutations cause measurable but incomplete receptor activation in vitro; however, specific triple and quadruple mutant combinations were constructed that promote an agonist-bound conformation, as measured by heteronuclear single quantum coherence NMR, and lead to full receptor activation. Moreover, these mutations retain functionality when introduced into divergent family members, and can therefore be used to dissect individual receptor function in vivo, which has been problematic because of redundancy and family size. Expression of activated PYL2 in Arabidopsis seeds activates ABA signaling by a number of measures: modulation of ABA-regulated gene expression, induction of hyperdormancy, and suppression of ABA deficiency phenotypes in the aba2-1 mutant. Our results set the stage for systematic gain-of-function studies of PYR1 and related ABA receptors and reveal that, despite the large number of receptors, activation of a single receptor is sufficient to activate signaling in planta. PMID:22139369

Melatonin is a neurohormone involved in the regulation of both acute and chronic pain whose mechanism is still not completely understood. We have recently demonstrated that selective MT2 melatonin receptor partial agonists have antiallodynic properties in animal models of chronic neuropathic pain by modulating ON/OFF cells of the descending antinociceptive system. Here, we examined the antinociceptive properties of the selective MT2 melatonin receptor partial agonists N-{2-[(3-methoxyphenyl)phenylamino]ethyl}acetamide (UCM765) and N-{2-[(3-bromophenyl)-(4-fluorophenyl)amino]ethyl}acetamide (UCM924) in two animal models of acute and inflammatory pain: the hot-plate and formalin tests. UCM765 and UCM924 (5-40 mg/kg, s.c.) dose-dependently increased the temperature of the first hind paw lick in the hot-plate test, and decreased the total time spent licking the injected hind paw in the formalin test. Antinociceptive effects of UCM765 and UCM924 were maximal at the dose of 20mg/kg. At this dose, the effects of UCM765 and UCM924 were similar to those produced by 200 mg/kg acetaminophen in the hot-plate test, and by 3 mg/kg ketorolac or 150 mg/kg MLT in the formalin test. Notably, antinociceptive effects of the two MT2 partial agonists were blocked by the pre-treatment with the MT2 antagonist 4-phenyl-2-propionamidotetralin (4P-PDOT, 10 mg/kg) in both paradigms. These results demonstrate the antinociceptive properties of UCM765 and UCM924 in acute and inflammatory pain models and corroborate the concept that MT2 melatonin receptor may be a novel target for analgesic drug development. PMID:26162699

Opioid abuse and dependence have evolved into an international epidemic as a significant clinical and societal problem with devastating consequences. Repeated exposure to the opioid, for example morphine, can induce profound, long-lasting behavioral sensitization and physical dependence, which are thought to reflect neuroplasticity in neural circuitry. Central serotonin (5-HT) neurotransmission participates in the development of dependence on and the expression of withdrawal from morphine. Serotonin 5-HT(2C) receptor (5-HT(2C)R) agonists suppress psychostimulant nicotine or cocaine-induced behavioral sensitization and drug-seeking behavior; however, the impact of 5-HT(2C)R agonists on behaviors relevant to opioid abuse and dependence has not been reported. In the present study, the effects of 5-HT(2C)R activation on the behavioral sensitization and naloxone-precipitated withdrawal symptoms were examined in mice underwent repeated exposure to morphine. Male mice received morphine (10 mg/kg, s.c.) to develop behavioral sensitization. Lorcaserin, a 5-HT(2C)R agonist, prevented the induction and expression, but not the development, of morphine-induced behavioral sensitization. Another cohort of mice received increasing doses of morphine over a 7-day period to induce morphine-dependence. Pretreatment of lorcaserin, or the positive control clonidine (an alpha 2-adrenoceptor agonist), ameliorated the naloxone-precipitated withdrawal symptoms. SB 242084, a selective 5-HT(2C)R antagonist, prevented the lorcaserin-mediated suppression of behavioral sensitization and withdrawal. Chronic morphine treatment was associated with an increase in the expression of 5-HT(2C)R protein in the ventral tegmental area, locus coeruleus and nucleus accumbens. These findings suggest that 5-HT(2C)R can modulate behavioral sensitization and withdrawal in morphine-dependent mice, and the activation of 5-HT(2C)R may represent a new avenue for the treatment of opioid addiction. PMID:26432939

The function of brain serotonin-2C (5-HT{sub 2C}) receptors, including behavioral and neurochemical responses to 5-HT{sub 2C} agonist challenge, has been suggested to be abnormal in individuals with neuropsychiatric disorders. Thus, it is important to identify polymorphisms and functional variants within this gene. Using SSCP analysis, the authors identified a Cys{sub 23}-Ser{sub 23} substitution (designated 5-HT{sub 2Ccys} and 5-HT{sub 2Cser}) in the first hydrophobic region of the human 5-HT{sub 2C} receptor. Allele frequencies in unrelated Caucasians were 0.13 and 0.87 for 5-HT{sub 2Cser} and 5-HT{sub 2Ccys}, respectively. DNAs from informative CEPH families were typed for this polymorphism and analyzed with respect to 20 linked markers on the X chromosome. Linkage analysis placed the 5-HT{sub 2C} receptor gene (HTR2C) on Xq24. To evaluate whether this amino acid substitution causes a variant function of this receptor, recombinant human 5-HT{sub 2Ccys} and 5-HT{sub 2Cser} receptors were expressed in Xenopus oocytes and tested for responses to 5-HT using electrophysiological techniques. Concentration-response curves for 5-HT were not significantly different in oocytes expressing either form of the receptor, suggesting that the 5-HT{sub 2Ccys} and 5-HT{sub 2Cser} receptor proteins may not differ in their responses to serotonin under baseline physiological conditions. 43 refs., 3 figs., 1 tab.

BACKGROUND AND PURPOSE Some agonists of ghrelin receptors cause rapid decreases in BP. The mechanisms by which they cause hypotension and the pharmacology of the receptors are unknown. EXPERIMENTAL APPROACH The effects of ligands of ghrelin receptors were investigated in rats in vivo, on isolated blood vessels and on cells transfected with the only molecularly defined ghrelin receptor, growth hormone secretagogue receptor 1a (GHSR1a). KEY RESULTS Three agonists of GHSR1a receptors, ulimorelin, capromorelin and CP464709, caused a rapid decrease in BP in the anaesthetized rat. The effect was not reduced by either of two GHSR1a antagonists, JMV2959 or YIL781, at doses that blocked effects on colorectal motility, in vivo. The rapid hypotension was not mimicked by ghrelin, unacylated ghrelin or the unacylated ghrelin receptoragonist, AZP531. The early hypotension preceded a decrease in sympathetic nerve activity. Early hypotension was not reduced by hexamethonium or by baroreceptor (sino-aortic) denervation. Ulimorelin also relaxed isolated segments of rat mesenteric artery, and, less potently, relaxed aorta segments. The vascular relaxation was not reduced by JMV2959 or YIL781. Ulimorelin, capromorelin and CP464709 activated GHSR1a in transfected HEK293 cells at nanomolar concentrations. JMV2959 and YIL781 both antagonized effects in these cells, with their pA2 values at the GHSR1a receptor being 6.55 and 7.84. CONCLUSIONS AND IMPLICATIONS Our results indicate a novel vascular receptor or receptors whose activation by ulimorelin, capromorelin and CP464709 lowered BP. This receptor is activated by low MW GHSR1a agonists, but is not activated by ghrelin. PMID:24670149

Activation of the serotonin (5-hydroxytryptamine, 5-HT) 5HT2C G protein-coupled receptor (GPCR) is proposed as novel pharmacotherapy for obesity and neuropsychiatric disorders. In contrast, activation of the 5-HT2A and 5-HT2B GPCRs is associated with untoward hallucinogenic and cardiopulmonary effects, respectively. There is no crystal structure available to guide design of 5-HT2Creceptor-specific ligands. For this reason, a homology model of the 5-HT2Creceptor was built based on the crystal structure of the human β2 adrenoceptor GPCR to delineate molecular determinants of ligand–receptor interactions for drug design purposes. Computational and experimental studies were carried out to validate the model. Binding of N(CH3)2-PAT [(1R, 3S)-(−)-trans-1-phenyl-3-N,N-dimethylamino-1,2,3,4-tetrahydronaphthalene], a novel 5-HT2Cagonist/5-HT2A/2B inverse agonist, and its secondary [NH(CH3)-PAT] and primary (NH2-PAT) amine analogs were studied at the 5-HT2C wild type (WT) and D3.32A, S3.36A, and Y7.43A 5-HT2C point-mutated receptors. Reference ligands included the tertiary amines lisuride and mesulergine and the primary amine 5-HT. Modeling results indicated that 5-HT2C residues D3.32, S3.36, and Y7.43 play a role in ligand binding. Experimental ligand binding results with WT and point-mutated receptors confirmed the impact of D3.32, S3.36, and Y7.43 on ligand affinity. PMID:24244046

Ligand-dependent activation of the aryl hydrocarbon receptor (AhR) pathway leads to a diverse array of biological and toxicological effects. The best-studied ligands for the AhR include polycyclic and halogenated aromatic hydrocarbons, the most potent of which is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, as new AhR ligands are identified and characterized, their structural and physiochemical diversity continues to expand. Our identification of AhR agonists in crude extracts from diverse materials raises questions as to the magnitude and extent of human exposure to AhR ligands through normal daily activities. We have found that solvent extracts of newspapers from countries around the world stimulate the AhR signaling pathway. AhR agonist activity was observed for dimethyl sulfoxide (DMSO), ethanol, and water extracts of printed newspaper, unprinted virgin paper, and black printing ink, where activation of luciferase reporter gene expression was transient, suggesting that the AhR active chemical(s) was metabolically labile. DMSO and ethanol extracts also stimulated AhR transformation and DNA binding, and also competed with [(3)H]TCDD for binding to the AhR. In addition, DMSO extracts of printed newspaper induced cytochrome P450 1A associated 7-ethoxyresorufin-O-deethylase activity in zebrafish embryos in vivo. Although the responsible bioactive chemical(s) remain to be identified, our results demonstrate that newspapers and printing ink contain relatively potent metabolically labile agonists of the AhR. Given the large amount of recycling and reprocessing of newspapers throughout the world, release of these easily extractable AhR agonists into the environment should be examined and their potential effects on aquatic organisms assessed. PMID:18203687

Acute pharmacological elevation of serotonin (5-hydroxytryptamine; 5-HT) activity decreases operant responding for primary reinforcers, suggesting that 5-HT reduces incentive motivation. The mechanism by which 5-HT alters incentive motivation is unknown, but parallel evidence that 5-HT2Creceptoragonists also reduce responding for primary reinforcers implicates this receptor as a potential candidate. These experiments examined whether chronic and acute disruptions of serotonin transporter (SERT) activity altered incentive motivation, and whether the 5-HT2Creceptor mediated the effects of elevated 5-HT on behavior. To assess incentive motivation, we measured responding for three different reinforcers: a primary reinforcer (saccharin), a conditioned reinforcer (CRf), and an unconditioned sensory reinforcer (USRf). In the chronic condition, responding was compared between SERT knockout (SERT-KO) mice and their wild-type littermates. In the acute condition, responding was examined in wild-type mice following treatment with 10 or 20 mg/kg citalopram, or its vehicle. The ability of the selective 5-HT2C antagonist SB 242084 to prevent the effects of SERT-KO and citalopram on responding was subsequently examined. Both SERT-KO and citalopram reduced responding for saccharin, a CRf, and a USRf. Treatment with SB 242084 enhanced responding for a CRf and a USRf in SERT-KO mice and blocked the effects of citalopram on CRf and USRf responding. However, SB 242084 was unable to prevent the effects of SERT-KO or citalopram on responding for saccharin. These results support a powerful inhibitory function for 5-HT in the control of incentive motivation, and indicate that the 5-HT2Creceptor mediates these effects of 5-HT in a reinforcer-dependent manner. PMID:27125304

A conformationally restricted analog of a selective cyclopropane-bearing serotonin 2Cagonist was designed and synthesized. A 2,2-dimethyl-2,3-dihydrobenzofuran scaffold was investigated as a constrained variant of a biologically active isopropyl phenyl ether. Construction of the required dimethyl-2,3-dihydrobenzofuran intermediate began using a procedure that relied on a microwave-assisted alkylation reaction. The synthesis of the designed compound as its HCl salt is reported in a total of 12 steps and 17% overall yield. Biological evaluation revealed the constrained analog to be a selective serotonin 2Cagonist with modest potency. PMID:26120215

The myogenic differentiation of C2C12 myoblast cells is induced by the novel androgen receptor (AR) partial agonist, (17α,20E)-17,20-[(1-methoxyethylidene)bis-(oxy)]-3-oxo-19-norpregna-4,20-diene-21-carboxylic acid methyl ester (YK11), as well as by dihydrotestosterone (DHT). YK11 is a selective androgen receptor modulator (SARM), which activates AR without the N/C interaction. In this study, we further investigated the mechanism by which YK11 induces myogenic differentiation of C2C12 cells. The induction of key myogenic regulatory factors (MRFs), such as myogenic differentiation factor (MyoD), myogenic factor 5 (Myf5) and myogenin, was more significant in the presence of YK11 than in the presence of DHT. YK11 treatment of C2C12 cells, but not DHT, induced the expression of follistatin (Fst), and the YK11-mediated myogenic differentiation was reversed by anti-Fst antibody. These results suggest that the induction of Fst is important for the anabolic effect of YK11. PMID:23995658

TRAIL/Apo-2L is a member of the TNF superfamily first described as an apoptosis-inducing cytokine in 1995. Similar to TNF and Fas ligand, TRAIL induces apoptosis in caspase-dependent manner following TRAIL death receptor trimerization. Because tumor cells were shown to be particularly sensitive to this cytokine while normal cells/tissues proved to be resistant along with being able to synthesize and release TRAIL, it was rapidly appreciated that TRAIL likely served as one of our major physiologic weapons against cancer. In line with this, a number of research laboratories and pharmaceutical companies have attempted to exploit the ability of TRAIL to kill cancer cells by developing recombinant forms of TRAIL or TRAIL receptoragonists (e.g., receptor-specific mAb) for therapeutic purposes. In this review article we will describe the biochemical pathways used by TRAIL to induce different cell death programs. We will also summarize the clinical trials related to this pathway and discuss possible novel uses of TRAIL-related therapies. In recent years, the physiological importance of TRAIL has expanded beyond being a tumoricidal molecule to one critical for a number of clinical settings - ranging from infectious disease and autoimmunity to cardiovascular anomalies. We will also highlight some of these conditions where modulation of the TRAIL/TRAIL receptor system may be targeted in the future. PMID:26343199

Activation of CB2 has been demonstrated to induce directed immune cell migration. However, the ability of CB2 to act as a chemoattractant receptor in macrophages remains largely unexplored. Using a real-time chemotaxis assay and a panel of chemically diverse and widely used CB2 agonists, we set out to examine whether CB2 modulates primary murine macrophage chemotaxis. We report that of 12 agonists tested, only JWH133, HU308, L-759,656 and L-759,633 acted as macrophage chemoattractants. Surprisingly, neither pharmacological inhibition nor genetic ablation of CB2 had any effect on CB2 agonist-induced macrophage chemotaxis. As chemotaxis was pertussis toxin sensitive in both WT and CB2-/- macrophages, we concluded that a non-CB1/CB2, Gi/o-coupled GPCR must be responsible for CB2 agonist-induced macrophage migration. The obvious candidate receptors GPR18 and GPR55 could not mediate JWH133 or HU308-induced cytoskeletal rearrangement or JWH133-induced β-arrestin recruitment in cells transfected with either receptor, demonstrating that neither are the unidentified GPCR. Taken together our results conclusively demonstrate that CB2 is not a chemoattractant receptor for murine macrophages. Furthermore we show for the first time that JWH133, HU308, L-759,656 and L-759,633 have off-target effects of functional consequence in primary cells and we believe that our findings have wide ranging implications for the entire cannabinoid field. PMID:26033291

To accelerate the healing processes in wound repair, attempts have been repeatedly made to use growth factors including thrombin and its peptide fragments. Unfortunately, the employment of thrombin is limited because of its high liability and pro-inflammatory actions at high concentrations. Some cellular effects of thrombin in wound healing are mediated by the activation of protease activated receptor-1 (PAR-1). The thrombin receptoragonist peptide (TRAP:SFLLRN) activates this receptor and mimics the effects of thrombin, but TRAP is a relatively weak agonist. We speculated that the encapsulated peptide may be more effective for PAR-1 activation than nonimmobilized peptide and developed a novel method for TRAP encapsulation in hydrogel films based on natural and synthetic polymers. The effects of an encapsulated TRAP in composite poly(N-vinyl caprolactam)-calcium alginate (PVCL) hydrogel films were investigated in a mouse model of wound healing. On day 7 the wound sizes decreased by about 60% under TRAP-chitosan-containing PVCL films, as compared with control films without TRAP. In the case of TRAP-polylysine-containing films no significant decrease in wound sizes was found. The fibroblast/macrophage ratio increased under TRAP-containing films on day 3 and on day 7. The number of proliferating fibroblasts increased to 150% under TRAP-chitosan films on day 7 as compared with control films. The number of [3H]-thymidine labeled endothelial and epithelial cells in granulation tissues was also enhanced. Thus, the immobilized TRAP to PVCL-chitosan hydrogel films were found to promote wound healing following the stimulation of fibroblast and epithelial cell proliferation and neovascularization. Furthermore, TRAP was shown to inhibit the secretion of the inflammatory mediator PAF from stimulated rat peritoneal mast cells due to augmentation of NO release from the mast cells. The encapsulated TRAP is suggested to accelerate wound healing due to the anti-inflammatory effects

The ADP receptor P2Y12 belongs to the superfamily of G protein–coupled receptors (GPCRs), and its activation triggers platelet aggregation. Therefore, potent antagonists, such as clopidogrel, are of high clinical relevance in prophylaxis and treatment of thromboembolic events. P2Y12 displays an elevated basal activity in vitro, and as such, inverse agonists may be therapeutically beneficial compared with antagonists. Only a few inverse agonists of P2Y12 have been described. To expand this limited chemical space and improve understanding of structural determinants of inverse agonist-receptor interaction, this study screened a purine compound library for lead structures using wild-type (WT) human P2Y12 and 28 constitutively active mutants. Results showed that ATP and ATP derivatives are agonists at P2Y12. The potency at P2Y12 was 2-(methylthio)-ADP > 2-(methylthio)-ATP > ADP > ATP. Determinants required for agonistic ligand activity were identified. Molecular docking studies revealed a binding pocket for the ATP derivatives that is bordered by transmembrane helices 3, 5, 6, and 7 in human P2Y12, with Y105, E188, R256, Y259, and K280 playing a particularly important role in ligand interaction. N-Methyl-anthraniloyl modification at the 3′-OH of the 2′-deoxyribose leads to ligands (mant-deoxy-ATP [dATP], mant-deoxy-ADP) with inverse agonist activity. Inverse agonist activity of mant-dATP was found at the WT human P2Y12 and half of the constitutive active P2Y12 mutants. This study showed that, in addition to ADP and ATP, other ATP derivatives are not only ligands of P2Y12 but also agonists. Modification of the ribose within ATP can result in inverse activity of ATP-derived ligands. PMID:23093496

Rationale Many monoamine releasers are abused by humans and produce abuse-related facilitation of intracranial self-stimulation (ICSS) in rats. Facilitation of ICSS in rats can be limited by monoamine releaser-induced serotonin (5-HT) release, but receptors that mediate 5-HT effects of monoamine releasers are unknown. Objectives Investigate whether 5-HT2Creceptor activation is necessary for rate-decreasing effects produced in an ICSS procedure in rats by the 5-HT-selective monoamine releaser fenfluramine and the non-selective releasers napthylisopropylamine (PAL-287) and (+)-3,4-methylenedioxymethamphetamine ((+)-MDMA). Methods Adult male Sprague-Dawley rats with electrodes implanted in the medial forebrain bundle were trained to lever press for brain stimulation under a “frequency-rate” ICSS procedure. Effectiveness of the 5-HT2C antagonist SB 242,084 was evaluated to block rate-decreasing effects produced by (1) the 5-HT2Cagonist Ro 60-0175, (2) the 5-HT-selective releaser fenfluramine, and (3) the mixed-action dopamine (DA)/norepinephrine (NE)/5-HT releasers PAL-287 (1.0-5.6 mg/kg), and (+)-MDMA (1.0-3.2 mg/kg). For comparison, effectiveness of SB 242,084 to alter rate-decreasing effects of the kappa opioid receptoragonist U69,593 and rate-increasing effects of the DA>5-HT releaser amphetamine were also examined. Results SB 242,084 pretreatment blocked rate-decreasing effects of Ro 60-0175 and fenfluramine, but not the rate-decreasing effects of U69,593 or the rate-increasing effects of amphetamine. SB 242,084 blunted the rate-decreasing effects and enhanced expression of rate-increasing effects of PAL-287 and (+)-MDMA. Conclusions These data suggest that 5-HT2Creceptor activation contributes to rate-decreasing effects that are produced by selective and mixed-action 5-HT releasers in rats and that may oppose and limit the expression of abuse-related ICSS facilitation by these compounds. PMID:26041338

The efficacy of serotonergic pharmacotherapy indicates that serotonin (5-HT) plays a role in the treatment, if not the etiology, of obsessive-compulsive disorder (OCD). While some clinical evidence implicates 5-HT(2C) receptors in this disorder, a definitive function has yet to be validated. We hypothesized that 5-HT(2C) receptor knockout (KO) mice may display compulsive-like behavior. This paper describes characterization of several distinct, highly organized behaviors in mice lacking functional 5-HT(2C) receptors, which supports a compulsive-like syndrome.Compulsive-like behavior was assessed in male 5-HT(2C) receptor KO and wildtype (WT) mice. Chewing of non-nutritive clay, chewing patterns on plastic-mesh screens, and the frequency of head dipping were measured. 5-HT(2C) receptor KO mice chewed more clay, produced a distinct pattern of "neat" chewing of plastic screens and exhibited reduced habituation of head dipping activity compared to WT mice. We conclude that the 5-HT(2C) receptor null mutant mouse provides a promising model of compulsive behavior and a means to further explore the role of 5-HT in OCD. PMID:12782219

The estrogen receptor β (ERβ) is emerging as an important player in the physiology of the endocrine pancreas. We evaluated the role and antidiabetic actions of the ERβ selective agonist WAY200070 as an insulinotropic molecule. We demonstrate that WAY200070 enhances glucose-stimulated insulin secretion both in mouse and human islets. In vivo experiments showed that a single administration of WAY200070 leads to an increase in plasma insulin levels with a concomitant improved response to a glucose load. Two-week treatment administration increased glucose-induced insulin release and pancreatic β-cell mass and improved glucose and insulin sensitivity. In addition, streptozotocin-nicotinamide–induced diabetic mice treated with WAY200070 exhibited a significant improvement in plasma insulin levels and glucose tolerance as well as a regeneration of pancreatic β-cell mass. Studies performed in db/db mice demonstrated that this compound restored first-phase insulin secretion and enhanced pancreatic β-cell mass. We conclude that ERβ agonists should be considered as new targets for the treatment of diabetes. PMID:23349481

It is well established that the dopamine (DA) and serotonin (5-HT) systems have extensive and complex interactions. However, the effects of specific 5-HT receptoragonists on traditionally DA-related behaviors remain unclear. Our goal in these studies was to characterize the effects of 5-HT receptoragonists on measures of locomotor activity and vertical rearing. The SSRIs fluoxetine and citalopram produced significant decreases in locomotor activity and vertical rearing at the highest doses used with females significant more sensitive to citalopram. The 5-HT1A agonist 8-OH-DPAT and the 5-HT2Cagonist MK 212 significantly decreased activity in both male and female mice, with females more sensitive to 8-OH-DPAT. In contrast, the 5-HT1B agonist RU 24969 and the 5-HT2A agonist DOI both increased activity, with DOI exhibiting differential effects with regard to sex. Finally, the 5-HT3 agonist SR 57227 produced significant locomotor increases only in female mice at the lowest dose. The results of these experiments define locomotor profiles of several 5-HT agonists in male and female C57BL/6J mice, providing a foundation for further explorations of 5-HT receptor effects on activity. PMID:19698737

Patients with rare defects in the gene encoding proopiomelanocortin (POMC) have extreme early-onset obesity, hyperphagia, hypopigmentation, and hypocortisolism, resulting from the lack of the proopiomelanocortin-derived peptides melanocyte-stimulating hormone and corticotropin. In such patients, adrenal insufficiency must be treated with hydrocortisone early in life. No effective pharmacologic treatments have been available for the hyperphagia and obesity that characterize the condition. In this investigator-initiated, open-label study, two patients with proopiomelanocortin deficiency were treated with setmelanotide, a new melanocortin-4 receptoragonist. The patients had a sustainable reduction in hunger and substantial weight loss (51.0 kg after 42 weeks in Patient 1 and 20.5 kg after 12 weeks in Patient 2). PMID:27468060

Most children with immune thrombocytopenia (ITP) will have spontaneous remission regardless of therapy, while about 20% will go on to have chronic ITP. In those children with chronic ITP who need treatment, standard therapies for acute ITP may have adverse effects that complicate their long-term use. Thus, alternative treatment options are needed for children with chronic ITP. Thrombopoietin receptoragonists (TPO-RA) have been shown to be safe and efficacious in adults with ITP, and represent a new treatment option for children with chronic ITP. One TPO-RA, eltrombopag, is now approved for children. Clinical trials in children are ongoing and data are emerging on safety and efficacy. This review will focus on the physiology of TPO-RA, their clinical use in children, as well as the long-term safety issues that need to be considered when using these agents. PMID:26322297

The innate immune system detects pathogens by the presence of highly conserved pathogen-expressed molecules, which trigger host immune defenses. Toll-like receptor (TLR) 9 detects unmethylated CpG dinucleotides in bacterial or viral DNA, and can be stimulated for therapeutic applications with synthetic oligodeoxynucleotides containing immune stimulatory "CpG motifs." TLR9 activation induces both innate and adaptive immunity. The TLR9-induced innate immune activation can be applied in the prevention or treatment of infectious diseases, and the adaptive immune-enhancing effects can be harnessed for improving vaccines. This article highlights the current understanding of the mechanism of action of CpG oligodeoxynucleotides, and provides an overview of the preclinical data and early human clinical trial results, applying these TLR9 agonists in the field of infectious diseases. PMID:17607015

Farnesoid X receptors (FXRs) are nuclear hormone receptors expressed in high amounts in body tissues that participate in bilirubin metabolism including the liver, intestines, and kidneys. Bile acids (BAs) are the natural ligands of the FXRs. FXRs regulate the expression of the gene encoding for cholesterol 7 alpha-hydroxylase, which is the rate-limiting enzyme in BA synthesis. In addition, FXRs play a critical role in carbohydrate and lipid metabolism and regulation of insulin sensitivity. FXRs also modulate live growth and regeneration during liver injury. Preclinical studies have shown that FXR activation protects against cholestasis-induced liver injury. Moreover, FXR activation protects against fatty liver injury in animal models of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH), and improved hyperlipidemia, glucose intolerance, and insulin sensitivity. Obeticholic acid (OCA), a 6α-ethyl derivative of the natural human BA chenodeoxycholic acid (CDCA) is the first-in-class selective FXR agonist that is ~100-fold more potent than CDCA. Preliminary human clinical trials have shown that OCA is safe and effective. In a phase II clinical trial, administration of OCA was well-tolerated, increased insulin sensitivity and reduced markers of liver inflammation and fibrosis in patients with type II diabetes mellitus and NAFLD. In two clinical trials of OCA in patients with primary biliary cirrhosis (PBC), a progressive cholestatic liver disease, OCA significantly reduced serum alkaline phosphatase (ALP) levels, an important disease marker that correlates well with clinical outcomes of patients with PBC. Together, these studies suggest that FXR agonists could potentially be used as therapeutic tools in patients suffering from nonalcoholic fatty and cholestatic liver diseases. Larger and Longer-term studies are currently ongoing. PMID:25705637

Farnesoid X receptors (FXRs) are nuclear hormone receptors expressed in high amounts in body tissues that participate in bilirubin metabolism including the liver, intestines, and kidneys. Bile acids (BAs) are the natural ligands of the FXRs. FXRs regulate the expression of the gene encoding for cholesterol 7 alpha-hydroxylase, which is the rate-limiting enzyme in BA synthesis. In addition, FXRs play a critical role in carbohydrate and lipid metabolism and regulation of insulin sensitivity. FXRs also modulate live growth and regeneration during liver injury. Preclinical studies have shown that FXR activation protects against cholestasis-induced liver injury. Moreover, FXR activation protects against fatty liver injury in animal models of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH), and improved hyperlipidemia, glucose intolerance, and insulin sensitivity. Obeticholic acid (OCA), a 6α-ethyl derivative of the natural human BA chenodeoxycholic acid (CDCA) is the first-in-class selective FXR agonist that is ~100-fold more potent than CDCA. Preliminary human clinical trials have shown that OCA is safe and effective. In a phase II clinical trial, administration of OCA was well-tolerated, increased insulin sensitivity and reduced markers of liver inflammation and fibrosis in patients with type II diabetes mellitus and NAFLD. In two clinical trials of OCA in patients with primary biliary cirrhosis (PBC), a progressive cholestatic liver disease, OCA significantly reduced serum alkaline phosphatase (ALP) levels, an important disease marker that correlates well with clinical outcomes of patients with PBC. Together, these studies suggest that FXR agonists could potentially be used as therapeutic tools in patients suffering from nonalcoholic fatty and cholestatic liver diseases. Larger and Longer-term studies are currently ongoing. PMID:25705637

Eye blinking is a spontaneous behavior observed in all mammals, and has been used as a well-established clinical indicator for dopamine production in neuropsychiatric disorders, including Parkinson's disease and Tourette syndrome [1,2]. Pharmacological studies in humans and non-human primates have shown that dopamine agonists/antagonists increase/decrease eye blinking rate. Common marmosets (Callithrix jacchus) have recently attracted a great deal of attention as suitable experimental animals in the psychoneurological field due to their more developed prefrontal cortex than rodents, easy handling compare to other non-human primates, and requirement for small amounts of test drugs. In this study, we evaluated the effects of dopamine D1-4 receptorsagonists on eye blinking in common marmosets. Our results show that the dopamine D1 receptoragonist SKF-82958 and the non-selective dopamine receptoragonist apomorphine significantly increased common marmosets eye blinking count, whereas the dopamine D2 agonist (+)-PHNO and the dopamine D3 receptoragonist (+)-PD-128907 produced somnolence in common marmosets resulting in a decrease in eye blinking count. The dopamine D4 receptoragonists PD-168077 and A-41297 had no effect on common marmosets' eye blinking count. Finally, the dopamine D1 receptor antagonist SCH 39166 completely blocked apomorphine-induced increase in eye blinking count. These results indicate that eye blinking in common marmosets may be a useful tool for in vivo screening of novel dopamine D1 receptoragonists as antipsychotics. PMID:26675887

The molecular mechanism(s) underlying cross-tolerance between mu and opioid receptor-like 1 (ORL1) receptoragonists were investigated using two human neuroblastoma cell lines endogenously expressing these receptors and G protein-coupled receptor kinases (GRKs). Prolonged (24 h) activation of the mu receptor desensitized both mu and ORL1 receptor-mediated inhibition of forskolin-stimulated cAMP accumulation and upregulated GRK2 levels in SH-SY5Y and BE(2)-C cells. Prolonged ORL1 activation increased GRK2 levels and desensitized both receptors in SH-SY5Y cells. Upregulation of GRK2 correlated with increases in levels of transcription factors Sp1 or AP-2. PD98059, an upstream inhibitor of extracellular signal-regulated kinases 1 and 2 (ERK1/2), reversed all these events. Pretreatment with orphanin FQ/nociceptin (OFQ/N) also upregulated GRK3 levels in both cell lines, and desensitized both receptors in BE(2)-C cells. Protein kinase C (PKC), but not ERK1/2, inhibition blocked OFQ/N-mediated GRK3 induction and mu and ORL1 receptor desensitization in BE(2)-C cells. Antisense DNA treatment confirmed the involvement of GRK2/3 in mu and ORL1 desensitization. Here, we demonstrate for the first time a role for ERK1/2-mediated GRK2 induction in the development of tolerance to mu agonists, as well as cross-tolerance to OFQ/N. We also demonstrate that chronic OFQ/N-mediated desensitization of ORL1 and mu receptors occurs via cell-specific pathways, involving ERK1/2-dependent GRK2, or PKC-dependent and ERK1/2-independent GRK3 induction. PMID:12423667

Isoteoline is a compound of aporphine structure derived from the alkaloid glaucine. Previous studies with isoteoline have shown antagonistic activity at 5-HT(2C) serotonergic receptors. We have investigated whether isoteoline interacts with 5-HT(1B) receptors. An isolation-induced social behavioural deficit test in mice was used as a model of stimulation of these receptors. The deficit in the behaviour of isolated mice in this experimental procedure was reported to be sensitive to 5-HT(1B)-receptor stimulation, since agonists at these receptors are capable of reversing it. In our study, we used N-(3-trifluoromethylphenyl)piperazine (TFMPP) (2 mg kg(-1)) as a reference agonist at these receptor sites. TFMPP completely restored the normal behaviour of the isolated mice. Its effect was prevented by propranolol (4 mg kg(-1)), a beta-adrenergic receptor antagonist with a high affinity for 5-HT(1B) receptors, which was inactive by itself. When isoteoline was given before TFMPP, it did not prevent the effect of the latter. Given alone at doses of 0.25, 1, 4 or 8 mg kg(-1), isoteoline showed an effect of its own to normalize the behaviour of isolated mice. The effect of isoteoline (1 mg kg(-1), i.p.) was antagonized by pretreatment with propranolol, indicating that it was mediated through stimulation of 5-HT(1B) receptors. Repeated treatment with isoteoline (1 mg kg(-1), 2 x 3 days, i.p.) produced tolerance to its effect and significantly attenuated the effect of TFMPP, when animals were tested 16 h after the last injection. In conclusion, the results provided functional evidence of agonist-like activity of isoteoline at the 5-HT(1B) receptors. PMID:12625876

The serotonin 2Creceptor regulates food uptake, and its activity is regulated by alternative pre-mRNA splicing. Alternative exon skipping is predicted to generate a truncated receptor protein isoform, whose existence was confirmed with a new antiserum. The truncated receptor sequesters the full-length receptor in intracellular membranes. We developed an oligonucleotide that promotes exon inclusion, which increases the ratio of the full-length to truncated receptor protein. Decreasing the amount of truncated receptor results in the accumulation of full-length, constitutively active receptor at the cell surface. After injection into the third ventricle of mice, the oligonucleotide accumulates in the arcuate nucleus, where it changes alternative splicing of the serotonin 2Creceptor and increases pro-opiomelanocortin expression. Oligonucleotide injection reduced food intake in both wild-type and ob/ob mice. Unexpectedly, the oligonucleotide crossed the blood-brain barrier and its systemic delivery reduced food intake in wild-type mice. The physiological effect of the oligonucleotide suggests that a truncated splice variant regulates the activity of the serotonin 2Creceptor, indicating that therapies aimed to change pre-mRNA processing could be useful to treat hyperphagia, characteristic for disorders like Prader-Willi syndrome. PMID:27406820

Background Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptors superfamily and are transcription factors activated by specific ligands. Liver X receptors (LXR) belong to the nuclear hormone receptors and have been shown to play an important role in cholesterol homeostasis. From the previous screening of several medicinal plants for potential partial PPARγ agonists, the extracts of Cornus alternifolia were found to exhibit promising bioactivity. In this paper, we report the isolation and structural elucidation of four new compounds and their potential as ligands for PPAR. Methods The new compounds were extracted from the leaves of Cornus alternifolia and fractionated by high-performance liquid chromatography. Their structures were elucidated on the basis of spectroscopic evidence and analysis of their hydrolysis products. Results Three new iridoid glycosides including an iridolactone, alternosides A-C (1–3), a new megastigmane glycoside, cornalternoside (4) and 10 known compounds, were obtained from the leaves of Cornus alternifolia. Kaempferol-3-O-β-glucopyranoside (5) exhibited potent agonistic activities for PPARα, PPARγ and LXR with EC50 values of 0.62, 3.0 and 1.8 μ M, respectively. Conclusions We isolated four new and ten known compounds from Cornus alternifolia, and one known compound showed agonistic activities for PPARα, PPARγ and LXR. General significance Compound 1 is the first example of a naturally occurring iridoid glycoside containing a β-glucopyranoside moiety at C-6. PMID:22353334

The actions of two morphine derivatives with short peptide side chains were evaluated upon the contraction of the isolated mouse vas deferens and upon displacement of /sup 3/H-etorphine from rat brain membranes. NIH-9833 (N-(6,14-endoetheno-7,8-dihydromorphine-7-alpha-carbonyl)-L-phenylalanyl-L-leucine ethyl ester HCl) was a potent agonist upon the vas deferens. Its EC50 for inhibition of the twitch was 1.2 +/- 0.1 nM. Both naltrexone (10/sup -7/ M) a relatively nonselective opioid antagonist, and ICI-174864 (10/sup -/' M) a highly selective delta receptor antagonist, blocked the actions of NIH-9833 which indicates that this drug is a delta receptoragonist. In contrast, NIH-9835 (N-(6,14-endoetheno-7,8-dihydromorphine-7-alpha-carbonyl)-L-glycyl-L-phenylalanyl-L-leucine ethyl ester HCl), which differs from NIH-9835 by the presence of a single amino acid residue, was devoid of opioid agonistic activity but was a potent antagonist of the inhibitory actions on the vas deferens of morphine and sufentanil. NIH-9833 and NIH-9835 were potent displacers of /sup 3/H-etorphine from rat cerebral membranes with EC50's of 0.58 nM and 1.7 nM, respectively. The observation that addition of a single glycyl group changes a dihydromorphine-peptide analog from a potent delta receptoragonist to an equally potent mu receptor antagonist suggests that the two receptor sites might be structurally quite similar.

Glutamate NMDA (N-methyl-D-aspartate) receptors are widely distributed in the central nervous system where they are involved in cognitive processes, motor control and many other functions. They are also well studied in the retina, which may be regarded as a biological model of the nervous system. However, little is known about NR2C and NR2D subunits of NMDA receptors, which have some specific features as compared to other subunits. Consequently the aim of the present study was to investigate their distribution in frog (Rana ridibunda) and turtle (Emys orbicularis) retinas which possess mixed and cone types of retina respectively. The experiments were performed using an indirect immunofluorescence method. Four antibodies directed to NR2C and NR2D subunits of NMDA receptor, as well as three antibodies directed to different splice variants of NR1 subunit, which is known to be obligatory for proper functioning of the receptor, were applied. All antibodies caused well expressed labeling in frog and turtle retinas. The NR2C and NR2D subunits were localized in glial Müller cells, while the NR1 subunit had both neuronal and glial localization. Our results show that glial NMDA receptors differ from neuronal ones in their subunit composition. The functional significance of the NMDA receptors and their NR2C and NR2D subunits, in particular for the neuron-glia interactions, is discussed. PMID:22386206

We mutated key amino acids of the human variant of the M1 muscarinic receptor that target ligand binding, receptor activation, and receptor-G protein interaction. We compared the effects of these mutations on the action of two atypical M1 functionally preferring agonists (N-desmethylclozapine and xanomeline) and two classical non-selective orthosteric agonists (carbachol and oxotremorine). Mutations of D105 in the orthosteric binding site and mutation of D99 located out of the orthosteric binding site decreased affinity of all tested agonists that was translated as a decrease in potency in accumulation of inositol phosphates and intracellular calcium mobilization. Mutation of D105 decreased the potency of the atypical agonist xanomeline more than that of the classical agonists carbachol and oxotremorine. Mutation of the residues involved in receptor activation (D71) and coupling to G-proteins (R123) completely abolished the functional responses to both classical and atypical agonists. Our data show that both classical and atypical agonists activate hM1 receptors by the same molecular switch that involves D71 in the second transmembrane helix. The principal difference among the studied agonists is rather in the way they interact with D105 in the orthosteric binding site. Furthermore, our data demonstrate a key role of D105 in xanomeline wash-resistant binding and persistent activation of hM1 by wash-resistant xanomeline. PMID:25882246

In keeping with its ability to control the mesoaccumbens dopamine (DA) pathway, the serotonin2Creceptor (5-HT2C R) plays a key role in mediating the behavioral and neurochemical effects of drugs of abuse. Studies assessing the influence of 5-HT2C R agonists on cocaine-induced responses have suggested that 5-HT2C Rs can modulate mesoaccumbens DA pathway activity independently of accumbal DA release, thereby controlling DA transmission in the nucleus accumbens (NAc). In the present study, we assessed this hypothesis by studying the influence of the 5-HT2C R agonist Ro 60-0175 on cocaine-induced behavioral, neurochemical and molecular responses. The i.p. administration of 1 mg/kg Ro 60-0175 inhibited hyperlocomotion induced by cocaine (15 mg/kg, i.p.), had no effect on cocaine-induced DA outflow in the shell, and increased it in the core subregion of the NAc. Furthermore, Ro 60-0175 inhibited the late-onset locomotion induced by the subcutaneous administration of the DA-D2 R agonist quinpirole (0.5 mg/kg), as well as cocaine-induced increase in c-Fos immunoreactivity in NAc subregions. Finally, Ro 60-0175 inhibited cocaine-induced phosphorylation of the DA and c-AMP regulated phosphoprotein of Mr 32 kDa (DARPP-32) at threonine residues in the NAc core, this effect being reversed by the selective 5-HT2C R antagonist SB 242084 (0.5 mg/kg, i.p.). Altogether, these findings demonstrate that 5-HT2C Rs are capable of modulating mesoaccumbens DA pathway activity at post-synaptic level by specifically controlling DA signaling in the NAc core subregion. In keeping with the tight relationship between locomotor activity and NAc DA function, this interaction could participate in the inhibitory control of cocaine-induced locomotor activity. PMID:24661380

The constitutive androstane receptor (CAR, NR1I3) is a key regulator of xenobiotic and endobiotic metabolism. The ligand-binding domains of murine (m) and human (h) CAR are divergent relative to other nuclear hormone receptors, resulting in species-specific differences in xenobiotic responses. Here we identify the widely used antiemetic meclizine (Antivert; Bonine) as both an agonist ligand for mCAR and an inverse agonist for hCAR. Meclizine increases mCAR transactivation in a dose-dependent manner. Like the mCAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, meclizine stimulates binding of steroid receptor coactivator 1 to the murine receptor in vitro. Meclizine administration to mice increases expression of CAR target genes in a CAR-dependent manner. In contrast, meclizine suppresses hCAR transactivation and inhibits the phenobarbital-induced expression of the CAR target genes, cytochrome p450 monooxygenase (CYP)2B10, CYP3A11, and CYP1A2, in primary hepatocytes derived from mice expressing hCAR, but not mCAR. The inhibitory effect of meclizine also suppresses acetaminophen-induced liver toxicity in humanized CAR mice. These results demonstrate that a single compound can induce opposite xenobiotic responses via orthologous receptors in rodents and humans. PMID:15272053

In vivo effects of GABAB receptor-positive modulators suggest that they have therapeutic potential for treating central nervous system disorders such as anxiety, depression, and drug abuse. Although these effects generally are thought to be mediated by positive modulation of GABAB receptors, such modulation has been examined primarily in vitro. The present study was aimed at further examining the in vivo positive modulatory properties of the GABAB receptor-positive modulators, 2,6-di-tert-butyl-4-(3-hydroxy-2,2-dimethylpropyl) phenol (CGP7930) and (R,S)-5,7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one (rac-BHFF). Both compounds enhanced loss of righting induced by baclofen in mice. However, CGP7930 was less effective and rac-BHFF was less potent for enhancing loss of righting induced by γ-hydroxybutyrate (GHB), which, like baclofen, has GABAB receptoragonist properties. In contrast with baclofen- and GHB-induced loss of righting, the hypothermic effects of baclofen and GHB were not enhanced by rac-BHFF but were enhanced by CGP7930 only at doses that produced hypothermia when given alone. CGP7930-induced hypothermia was not attenuated by the GABAB receptor antagonist 3-aminopropyl(diethoxymethyl)phosphinic acid (CGP35348), at doses that blocked baclofen-induced hypothermia, and was not increased by the nitric-oxide synthase inhibitor Nω-nitro-l-arginine methyl ester, at doses that increased the hypothermic effects of baclofen and GHB. The results provide evidence that CGP7930 and rac-BHFF act in vivo as positive modulators at GABAB receptors mediating loss of righting, but not at GABAB receptors mediating hypothermia. Conceivably, CGP7930, but not rac-BHFF, acts as an allosteric agonist at these latter receptors. Taken together, the results provide further evidence of pharmacologically distinct GABAB receptor subtypes, possibly allowing for a more selective therapeutic interference with the GABAB system. PMID:20628000

Modification of a phenolic lead structure based on lessons learned from increasing the potency of steroidal glucocorticoid agonists lead to the discovery of exceptionally potent, nonsteroidal, indazole GR agonists. SAR was developed to achieve good selectivity against other nuclear hormone receptors with the ultimate goal of achieving a dissociated GR agonist as measured by human in vitro assays. The specific interactions by which this class of compounds inhibits GR was elucidated by solving an X-ray co-crystal structure. PMID:23953070

G protein-coupled receptor 119 (GPR119) is involved in regulating metabolic homoeostasis, with GPR119 agonists targeted for the treatment of type-2 diabetes and obesity. Using the endogenous agonist oleoylethanolamide and a number of small molecule synthetic agonists we have investigated the temporal dynamics of receptor signalling. Using both a dynamic luminescence biosensor-based assay and an endpoint cAMP accumulation assay we show that agonist-driven desensitization is not a major regulatory mechanism for GPR119 despite robust activation responses, regardless of the agonist used. Temporal analysis of the cAMP responses demonstrated sustained signalling resistant to washout for some, but not all of the agonists tested. Further analysis indicated that the sustained effects of one synthetic agonist AR-231,453 were consistent with a role for slow dissociation kinetics. In contrast, the sustained responses to MBX-2982 and AZ1 appeared to involve membrane deposition. We also detect wash-resistant responses to AR-231,453 at the level of physiologically relevant responses in an endogenous expression system (GLP-1 secretion in GLUTag cells). In conclusion, our findings indicate that in a recombinant expression system GPR119 activation is sustained, with little evidence of pronounced receptor desensitization, and for some ligands persistent agonist responses continue despite removal of excess agonist. This provides novel understanding of the temporal responses profiles of potential drug candidates targetting GPR119, and highlights the importance of carefully examining the the mechanisms through which GPCRs generate sustained responses. PMID:26101059

Glucagon-like peptide-1 (GLP-1) is a member of the proglucagon incretin family, and GLP-1 receptoragonists (RAs) have been introduced as a new class of antidiabetic medications in the past decade. The benefits of GLP-1 RAs are derived from their pleiotropic effects, which include glucose-dependent insulin secretion, suppressed glucagon secretion, and reduced appetite. Moreover, GLP-1 RAs also exert beneficial roles on multiple organ systems in which the GLP-1 receptors exist, including the cardiovascular system. Cardiovascular effects of GLP-1 RAs have been of great interest since the burden from cardiovascular diseases (CVD) has been unbearably increasing in a diabetic population worldwide, despite strict glycemic control and advanced therapeutic techniques to treat CVD. Preclinical studies have already demonstrated the beneficial effects of GLP-1 on myocardium and vascular endothelium, and many clinical studies evaluating changes in surrogate markers of CVD have suggested potential benefits from the use of GLP-1 RAs. Data from numerous clinical trials primarily evaluating the antihyperglycemic effects of multiple GLP-1 RAs have also revealed that changes in most CVD risk markers reported as secondary outcomes have been in favor of GLP-1 RAs treatment. However, to date, there is only one randomized clinical trial of GLP-1 RAs (the ELIXA study) evaluating major cardiovascular events as their primary outcomes, and in this study, a neutral cardiovascular effect of lixisenatide was observed in high-risk diabetic subjects. Therefore, the results of ongoing CVD outcome trials with the use of GLP-1 RAs should be awaited to elucidate the translation of benefits previously seen in CVD risk marker studies into large clinical trials with primary cardiovascular outcomes. PMID:27118277

Glucagon-like peptide-1 (GLP-1) is a member of the proglucagon incretin family, and GLP-1 receptoragonists (RAs) have been introduced as a new class of antidiabetic medications in the past decade. The benefits of GLP-1 RAs are derived from their pleiotropic effects, which include glucose-dependent insulin secretion, suppressed glucagon secretion, and reduced appetite. Moreover, GLP-1 RAs also exert beneficial roles on multiple organ systems in which the GLP-1 receptors exist, including the cardiovascular system. Cardiovascular effects of GLP-1 RAs have been of great interest since the burden from cardiovascular diseases (CVD) has been unbearably increasing in a diabetic population worldwide, despite strict glycemic control and advanced therapeutic techniques to treat CVD. Preclinical studies have already demonstrated the beneficial effects of GLP-1 on myocardium and vascular endothelium, and many clinical studies evaluating changes in surrogate markers of CVD have suggested potential benefits from the use of GLP-1 RAs. Data from numerous clinical trials primarily evaluating the antihyperglycemic effects of multiple GLP-1 RAs have also revealed that changes in most CVD risk markers reported as secondary outcomes have been in favor of GLP-1 RAs treatment. However, to date, there is only one randomized clinical trial of GLP-1 RAs (the ELIXA study) evaluating major cardiovascular events as their primary outcomes, and in this study, a neutral cardiovascular effect of lixisenatide was observed in high-risk diabetic subjects. Therefore, the results of ongoing CVD outcome trials with the use of GLP-1 RAs should be awaited to elucidate the translation of benefits previously seen in CVD risk marker studies into large clinical trials with primary cardiovascular outcomes. PMID:27118277

Desensitization of µ-opioid receptors (MORs) develops over 5–15 minutes after the application of some, but not all, opioid agonists and lasts for tens of minutes after agonist removal. The decrease in function is receptor selective (homologous) and could result from 1) a reduction in receptor number or 2) a decrease in receptor coupling. The present investigation used photolysis of two caged opioid ligands to examine the kinetics of MOR-induced potassium conductance before and after MOR desensitization. Photolysis of a caged antagonist, carboxynitroveratryl-naloxone (caged naloxone), blocked the current induced by a series of agonists, and the time constant of decline was significantly decreased after desensitization. The increase in the rate of current decay was not observed after partial blockade of receptors with the irreversible antagonist, β-chlornaltrexamine (β-CNA). The time constant of current decay after desensitization was never more rapid than 1 second, suggesting an increased agonist off-rate rather than an increase in the rate of channel closure downstream of the receptor. The rate of G protein–coupled K+ channel (GIRK) current activation was examined using photolysis of a caged agonist, carboxynitrobenzyl-tyrosine-[Leu5]-enkephalin. After acute desensitization or partial irreversible block of MORs with β-CNA, there was an increase in the time it took to reach a peak current. The decrease in the rate of agonist-induced GIRK conductance was receptor selective and dependent on receptor number. The results indicate that opioid receptor desensitization reduced the number of functional receptor and that the remaining active receptors have a reduced agonist affinity. PMID:24748657

Fluorescence spectroscopic studies are powerful tools for the evaluation of receptor structure and the dynamic changes associated with receptor activation. Here, we have developed two chemically distinct fluorescent probes of the cholecystokinin (CCK) receptor by attaching acrylodan or a nitrobenzoxadiazole moiety to the amino terminus of a partial agonist CCK analogue. These two probes were able to bind to the CCK receptor specifically and with high affinity, and were able to elicit only submaximal intracellular calcium responses typical of partial agonists. The fluorescence characteristics of these probes were compared with those previously reported for structurally-related full agonist and antagonist probes. Like the previous probes, the partial agonist probes exhibited longer fluorescence lifetimes and increased anisotropy when bound to the receptor than when free in solution. The receptor-bound probes were not easily quenched by potassium iodide, suggesting that the fluorophores were protected from the extracellular aqueous milieu. The fluorescence characteristics of the partial agonist probes were quite similar to those of the analogous full agonist probes and quite distinct from the analogous antagonist probes. These data suggest that the partially activated conformational state of this receptor is more closely related to its fully active state than to its inactive state. PMID:16779661

Background A homologue of the ecdysone receptor has previously been identified in human filarial parasites. As the ecdysone receptor is not found in vertebrates, it and the regulatory pathways it controls represent attractive potential chemotherapeutic targets. Methodology/ Principal Findings Administration of 20-hydroxyecdysone to gerbils infected with B. malayi infective larvae disrupted their development to adult stage parasites. A stable mammalian cell line was created incorporating the B. malayi ecdysone receptor ligand-binding domain, its heterodimer partner and a secreted luciferase reporter in HEK293 cells. This was employed to screen a series of ecdysone agonist, identifying seven agonists active at sub-micromolar concentrations. A B. malayi ecdysone receptor ligand-binding domain was developed and used to study the ligand-receptor interactions of these agonists. An excellent correlation between the virtual screening results and the screening assay was observed. Based on both of these approaches, steroidal ecdysone agonists and the diacylhydrazine family of compounds were identified as a fruitful source of potential receptoragonists. In further confirmation of the modeling and screening results, Ponasterone A and Muristerone A, two compounds predicted to be strong ecdysone agonists stimulated expulsion of microfilaria and immature stages from adult parasites. Conclusions The studies validate the potential of the B. malayi ecdysone receptor as a drug target and provide a means to rapidly evaluate compounds for development of a new class of drugs against the human filarial parasites. PMID:27300294

Decreasing the temperature to 30°C is accompanied by significant enhancement of α2C-AR plasma membrane levels in several cell lines with fibroblast phenotype, as demonstrated by radioligand binding in intact cells or isolated membranes. No changes were observed on the effects of low-temperature after blocking receptor internalization in α2C-AR transfected HEK293T cells. In contrast, two pharmacological chaperones, dimethyl sulfoxide and glycerol, increased the cell surface receptor levels at 37°C, but not at 30°C. Further, at 37°C α2C-AR is co-localized with endoplasmic reticulum markers, but not with the lysosomal markers. Treatment with three distinct HSP90 inhibitors, radicicol, macbecin and 17-DMAG significantly enhanced α2C-AR cell surface levels at 37°C, but these inhibitors had no effect at 30°C. Similar results were obtained after decreasing the HSP90 cellular levels using specific siRNA. Co-immunoprecipitation experiments demonstrated that α2C-AR interacts with HSP90 and this interaction is decreased at 30°C. The contractile response to endogenous α2C-AR stimulation in rat tail artery was also enhanced at reduced temperature. Similar to HEK293T cells, HSP90 inhibition increased the α2C-AR contractile effects only at 37°C. Moreover, exposure to low-temperature of vascular smooth muscle cells from rat tail artery decreased the cellular levels of HSP90, but did not change HSP70 levels. These data demonstrate that exposure to low-temperature augments the α2C-AR transport to the plasma membrane by releasing the inhibitory activity of HSP90 on the receptor traffic, findings which may have clinical relevance for the diagnostic and treatment of Raynaud Phenomenon. PMID:21145921

Reducing Na(+) in the extracellular environment may lead to two beneficial effects for increasing agonist binding to cell surface G-protein coupled receptors (GPCRs): reduction of Na(+)-mediated binding block and reduce of receptor internalization. However, such combined effects have not been explored. We used Chinese Hamster Ovary cells expressing vasopressin V1b receptors as a model to explore Na(+) sensitivity in agonist binding and receptor internalization. Under basal conditions, a large fraction of V1b receptors is located intracellularly, and a small fraction is in the plasma membrane. Decreases in external Na(+) increased cell surface [(3)H]AVP binding and decreased receptor internalization. Substitution of Na(+) by Cs(+) or NH4(+) inhibited agonist binding. To suppress receptor internalization, the concentration of NaCl, but not of CsCl, had to be less than 50 mM, due to the high sensitivity of the internalization machinery to Na(+) over Cs(+). Iso-osmotic supplementation of glucose or NH4Cl maintained internalization of the V1b receptor, even in a low-NaCl environment. Moreover, iodide ions, which acted as a counter anion, inhibited V1b agonist binding. In summary, we found external ionic conditions that could increase the presence of high-affinity state receptors at the cell surface with minimum internalization during agonist stimulations. PMID:27138239

Reducing Na+ in the extracellular environment may lead to two beneficial effects for increasing agonist binding to cell surface G-protein coupled receptors (GPCRs): reduction of Na+-mediated binding block and reduce of receptor internalization. However, such combined effects have not been explored. We used Chinese Hamster Ovary cells expressing vasopressin V1b receptors as a model to explore Na+ sensitivity in agonist binding and receptor internalization. Under basal conditions, a large fraction of V1b receptors is located intracellularly, and a small fraction is in the plasma membrane. Decreases in external Na+ increased cell surface [3H]AVP binding and decreased receptor internalization. Substitution of Na+ by Cs+ or NH4+ inhibited agonist binding. To suppress receptor internalization, the concentration of NaCl, but not of CsCl, had to be less than 50 mM, due to the high sensitivity of the internalization machinery to Na+ over Cs+. Iso-osmotic supplementation of glucose or NH4Cl maintained internalization of the V1b receptor, even in a low-NaCl environment. Moreover, iodide ions, which acted as a counter anion, inhibited V1b agonist binding. In summary, we found external ionic conditions that could increase the presence of high-affinity state receptors at the cell surface with minimum internalization during agonist stimulations. PMID:27138239

High throughput screening combined with efficient datamining and parallel synthesis led to the discovery of a novel series of indolines showing potent in vitro ghrelin receptoragonist activity and acceleration of gastric emptying in rats. PMID:17942309

As part of an on-going lead optimisation effort, a cross screening exercise identified an aryl sulphonyl amide hit that was optimised to afford a highly potent series of ghrelin receptoragonists. PMID:19128969

ABSTRACT Accumulating preclinical evidence indicates that Toll-like receptor (TLR) agonists efficiently boost tumor-targeting immune responses (re)initiated by most, if not all, paradigms of anticancer immunotherapy. Moreover, TLR agonists have been successfully employed to ameliorate the efficacy of various chemotherapeutics and targeted anticancer agents, at least in rodent tumor models. So far, only three TLR agonists have been approved by regulatory agencies for use in cancer patients. Moreover, over the past decade, the interest of scientists and clinicians in these immunostimulatory agents has been fluctuating. Here, we summarize recent advances in the preclinical and clinical development of TLR agonists for cancer therapy. PMID:27141345

Using the X-ray crystal structure of an amide-based progesterone receptor (PR) partial agonist bound to the PR ligand binding domain, a novel PR partial agonist class containing a pyrrolidine ring was designed. Members of this class of N-alkylpyrrolidines demonstrate potent and highly selective partial agonism of the progesterone receptor, and one of these analogs was shown to be efficacious upon oral dosing in the OVX rat model of estrogen opposition.

Brain nicotinic acetylcholine receptors are involved in several neuropsychiatric disorders, e.g. Alzheimer's and Parkinson's diseases, Tourette's syndrome, schizophrenia, depression, autism, attention deficit hyperactivity disorder, and anxiety. Currently, approaches selectively targeting the activation of specific nicotinic acetylcholine receptors are in clinical development for treatment of memory impairment of Alzheimer's disease patients. These are α4β2 and α7 nicotinic acetylcholine receptoragonists which are believed to enhance cholinergic and glutamatergic neurotransmission, respectively. In order to gain a better insight into the mechanistic role of these two nicotinic acetylcholine receptors in learning and memory, we investigated the effects of the α4β2 nicotinic acetylcholine receptoragonist TC-1827 and the α7 nicotinic acetylcholine receptor partial agonist SSR180711 on hippocampal long-term potentiation (LTP), a widely accepted cellular experimental model of memory formation. Generally, LTP is distinguished in an early and a late form, the former being protein-synthesis independent and the latter being protein-synthesis dependent. TC-1827 was found to increase early LTP in a bell-shaped dose dependent manner, but did not affect late LTP. In contrast, the α7 nicotinic acetylcholine receptor partial agonist SSR180711 showed enhancing effects on both early and late LTP in a bell-shaped manner. Furthermore, SSR180711 not only increased early LTP, but also transformed it into late LTP, which was not observed with the α4β2 nicotinic acetylcholine receptoragonist. Therefore, based on these findings α7 nicotinic acetylcholine receptor (partial) agonists appear to exhibit stronger efficacy on memory improvement than α4β2 nicotinic acetylcholine receptoragonists. PMID:21968142

The serotonin receptor2C (HTR2C) gene has been shown to play a pivotal role in major depression. We examined the association between post-stroke depression (PSD) and polymorphism in HTR2C. A cohort of 223 patients with acute lacunar stroke admitted to the stroke unit of a university-affiliated regional hospital in Hong Kong was recruited. Three months after the onset of the index stroke, a research assistant administered the locally validated 15-item Geriatric Depression Scale. PSD was defined as a geriatric depression scale score of 7 or above. Possible confounding factors, including previous history of stroke, severity of stroke, level of social support, and recent life events, were investigated. All patients were genotyped for polymorphisms of HTR2C. Separate analyses were performed for males and females. Sixty-one patients were found to have PSD. There were significant associations between the HTR2C gene and PSD status in the male patients, but not in the female ones. After adjusting for possible confounders, the rs12837651 T allele (odds ratio = 4.020) and the rs2192371 G allele (odds ratio = 2.866) were found to be significantly associated with PSD in males. Genetic variation in HTR2Creceptors appears to be involved in the pathogenesis of PSD in Chinese males. PMID:23765961

Polymorphisms in the gene encoding the serotonin synthesis enzyme Tph2 have been identified in mental illnesses, including bipolar disorder, major depression, autism, schizophrenia, and ADHD. Deficits in cognitive flexibility and perseverative behaviors are shared common symptoms in these disorders. However, little is known about the impact of Tph2 gene variants on cognition. Mice expressing a human TPH2 variant (Tph2-KI) were used to investigate cognitive consequences of TPH2 loss of function and pharmacological treatments. We applied a recently developed behavioral assay, the automated H-maze, to study cognitive functions in Tph2-KI mice. This assay involves the consecutive discovery of three different rules: a delayed alternation task, a non-alternation task, and a delayed reversal task. Possible contribution of locomotion, reward, and sensory perception were also investigated. The expression of loss-of-function mutant Tph2 in mice was associated with impairments in reversal learning and cognitive flexibility, accompanied by perseverative behaviors similar to those observed in human clinical studies. Pharmacological restoration of 5-HT synthesis with 5-hydroxytryptophan or treatment with the 5-HT2Creceptoragonist CP809.101 reduced cognitive deficits in Tph2-KI mice and abolished perseveration. In contrast, treatment with the psychostimulant methylphenidate exacerbated cognitive deficits in mutant mice. Results from this study suggest a contribution of TPH2 in the regulation of cognition. Furthermore, identification of a role for a 5-HT2 receptoragonist as a cognition-enhancing agent in mutant mice suggests a potential avenue to explore for the personalized treatment of cognitive symptoms in humans with reduced 5-HT synthesis and TPH2 polymorphisms. PMID:24196946

It is well known that allosteric modulators of muscarinic acetylcholine receptors can both diminish and increase the affinity of receptors for their antagonists. We investigated whether the allosteric modulators can also increase the affinity of receptors for their agonists. Twelve agonists and five allosteric modulators were tested in experiments on membranes of CHO cells that had been stably transfected with genes for the M1-M4 receptor subtypes. Allosterically induced changes in the affinities for agonists were computed from changes in the ability of a fixed concentration of each agonist to compete with [3H]N-methylscopolamine for the binding to the receptors in the absence and the presence of varying concentrations of allosteric modulators. The effects of allosteric modulators varied greatly depending on the agonists and the subtypes of receptors. The affinity for acetylcholine was augmented by (-)-eburnamonine on the M2 and M4 receptors and by brucine on the M1 and M3 receptors. Brucine also enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pilocarpine, 3-(3-pentylthio-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1- methylpyridine (pentylthio-TZTP), oxotremorine-M, and McN-A-343 on the M1, M3, and M4 receptors, for pentylthio-TZTP on the M2 receptors, and for arecoline on the M3 receptors. (-)-Eburnamonine enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pentylthio-TZTP, pilocarpine, oxotremorine and oxotremorine-M on the M2 receptors and for pilocarpine on the M4 receptors. Vincamine, strychnine, and alcuronium displayed fewer positive allosteric interactions with the agonists, but each allosteric modulator displayed positive cooperativity with at least one agonist on at least one muscarinic receptor subtype. The highest degrees of positive cooperativity were observed between (-)-eburnamonine and pilocarpine and (-)-eburnamonine and oxotremorine-M on the M2 receptors (25- and 7-fold increases in

Corneal transplantation is the most used therapy for eye disorders. Although the cornea is somewhat an immune privileged organ, immune rejection is still the major problem that reduces the success rate. Therefore, effective chemical drugs that regulate immunoreactions are needed to improve the outcome of corneal transplantations. Here, a sphingosine-1-phosphate receptor 1 (S1P1) selective agonist was systematically evaluated in mouse allogeneic corneal transplantation and compared with the commonly used immunosuppressive agents. Compared with CsA and the non-selective sphingosine 1-phosphate (S1P) receptoragonist FTY720, the S1P1 selective agonist can prolong the survival corneal transplantation for more than 30 days with a low immune response. More importantly, the optimal dose of the S1P1 selective agonist was much less than non-selective S1P receptoragonist FTY720, which would reduce the dose-dependent toxicity in drug application. Then we analyzed the mechanisms of the selected S1P1 selective agonist on the immunosuppression. The results shown that the S1P1 selective agonist could regulate the distribution of the immune cells with less CD4+ T cells and enhanced Treg cells in the allograft, moreover the expression of anti-inflammatory cytokines TGF-β1 and IL-10 unregulated which can reduce the immunoreactions. These findings suggest that S1P1 selective agonist may be a more appropriate immunosuppressive compound to effectively prolong mouse allogeneic corneal grafts survival. PMID:25216235

Timing deficits are observed in patients with schizophrenia. Serotonergic hallucinogens can also alter the subjective experience of time. Characterizing the mechanism through which the serotonergic system regulates timing will increase our understanding of the linkage between serotonin (5-HT) and schizophrenia, and will provide insight into the mechanism of action of hallucinogens. We investigated whether interval timing in mice is altered by hallucinogens and other 5-HT2 receptor ligands. C57BL/6J mice were trained to perform a discrete-trials temporal discrimination task. In the discrete-trials task, mice were presented with two levers after a variable interval. Responding on lever A was reinforced if the interval was <6.5 s, and responding on lever B was reinforced if the interval was >6.5 s. A 2-parameter logistic function was fitted to the proportional choice for lever B (%B responding), yielding estimates of the indifference point (T50) and the Weber fraction (a measure of timing precision). The 5-HT2A antagonist M100907 increased T50, whereas the 5-HT2C antagonist SB-242,084 reduced T50. The results indicate that 5-HT2A and 5-HT2Creceptors have countervailing effects on the speed of the internal pacemaker. The hallucinogen 2,5-dimethoxy-4-iodoamphetamine (DOI; 3 mg/kg IP), a 5-HT2 agonist, flattened the response curve at long stimulus intervals and shifted it to the right, causing both T50 and the Weber fraction to increase. The effect of DOI was antagonized by M100907 (0.03 mg/kg SC) but was unaffected by SB-242,084 (0.1 mg/kg SC). Similar to DOI, the selective 5-HT2A agonist 25CN-NBOH (6 mg/kg SC) reduced %B responding at long stimulus intervals, and increased T50 and the Weber fraction. These results demonstrate that hallucinogens alter temporal perception in mice, effects that are mediated by the 5-HT2A receptor. It appears that 5-HT regulates temporal perception, suggesting that altered serotonergic signaling may contribute to the timing deficits

NOR1, Nur77 and Nurr1 are orphan nuclear receptors and members of the NR4A subfamily. Here, we report that the expression of hypothalamic NOR1 was remarkably decreased in mildly obese {beta}-endorphin-deficient mice and obese db/db mice with the leptin receptor mutation, compared with age-matched wild-type mice, whereas there were no genotypic differences in the expression of hypothalamic Nur77 or Nurr1 in these animals. The injection of NOR1 siRNA oligonucleotide into the third cerebral ventricle significantly suppressed food intake and body weight in mice. On the other hand, the decreases in hypothalamic NOR1 expression were not found in non-obese 5-HT2Creceptor-deficient mice. Moreover, systemic administration of m-chlorophenylpiperazine (mCPP), a 5-HT2C/1B receptoragonist, had no effect on hypothalamic NOR1 expression, while suppressing food intake in {beta}-endorphin-deficient mice. These findings suggest that 5-HT2Creceptor-independent proopiomelanocortin-derived peptides regulate the expression of hypothalamic NOR1, which is a novel modulator of feeding behavior and energy balance.

The questions of whether G protein-coupled receptors exist as monomers, dimers, and/or oligomers and if these species interconvert in a ligand-dependent manner are among the most contentious current issues in biology. When employing spatial intensity distribution analysis to laser scanning confocal microscope images of cells stably expressing either a plasma membrane-associated form of monomeric enhanced green fluorescent protein (eGFP) or a tandem version of this fluorophore, the eGFP tandem was identified as a dimer. Similar studies on cells stably expressing an eGFP-tagged form of the epidermal growth factor receptor demonstrated that, although largely a monomer in the basal state, this receptor rapidly became predominantly dimeric upon the addition of its ligand epidermal growth factor. In cells induced to express an eGFP-tagged form of the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor, global analysis of construct quantal brightness was consistent with the predominant form of the receptor being dimeric. However, detailed spatial intensity distribution analysis demonstrated the presence of multiple forms ranging from monomers to higher-order oligomers. Furthermore, treatment with chemically distinct 5-HT2Creceptor antagonists resulted in a time-dependent change in the quaternary organization to one in which there was a preponderance of receptor monomers. This antagonist-mediated effect was reversible, because washout of the ligand resulted in the regeneration of many of the oligomeric forms of the receptor. PMID:25825490

G protein-coupled receptors (GPCRs) play a pivotal role in cell signalling, controlling many processes such as immunity, growth, cellular differentiation, neurological pathways and hormone secretions. Fatty acid agonists are increasingly recognised as having a key role in the regulation of glucose homoeostasis via stimulation of islet and gastrointestinal GPCRs. Downstream cell signalling results in modulation of the biosynthesis, secretion, proliferation and anti-apoptotic pathways of islet and enteroendocrine cells. GPR40 and GPR120 are activated by long-chain fatty acids (>C12) with both receptors coupling to the Gαq subunit that activates the Ca(2+)-dependent pathway. GPR41 and GPR43 are stimulated by short-chain fatty acids (C2-C5), and activation results in binding to Gαi that inhibits the adenylyl cyclase pathway attenuating cAMP production. In addition, GPR43 also couples to the Gαq subunit augmenting intracellular Ca(2+) and activating phospholipase C. GPR55 is specific for cannabinoid endogenous agonists (endocannabinoids) and non-cannabinoid fatty acids, which couples to Gα12/13 and Gαq proteins, leading to enhancing intracellular Ca(2+), extracellular signal-regulated kinase 1/2 (ERK) phosphorylation and Rho kinase. GPR119 is activated by fatty acid ethanolamides and binds to Gαs utilising the adenylate cyclase pathway, which is dependent upon protein kinase A. Current research indicates that GPCR therapies may be approved for clinical use in the near future. This review focuses on the recent advances in preclinical diabetes research in the signalling and regulation of GPCRs on islet and enteroendocrine cells involved in glucose homoeostasis. PMID:26739335

TAM-163, an agonist monoclonal antibody targeting tyrosine receptor kinase-B (TrkB), is currently being investigated as a potential body weight modulatory agent in humans. To support the selection of the dose range for the first-in-human (FIH) trial of TAM-163, we conducted a mechanistic analysis of the pharmacokinetic (PK) and pharmacodynamic (PD) data (e.g., body weight gain) obtained in lean cynomolgus and obese rhesus monkeys following single doses ranging from 0.3 to 60 mg/kg. A target-mediated drug disposition (TMDD) model was used to describe the observed nonlinear PK and Emax approach was used to describe the observed dose-dependent PD effect. The TMDD model development was supported by the experimental determination of the binding affinity constant (9.4 nM) and internalization rate of the drug-target complex (2.08 h−1). These mechanistic analyses enabled linking of exposure, target (TrkB) coverage, and pharmacological activity (e.g., PD) in monkeys, and indicated that ≥ 38% target coverage (time-average) was required to achieve significant body weight gain in monkeys. Based on the scaling of the TMDD model from monkeys to humans and assuming similar relationship between the target coverage and pharmacological activity between monkey and humans, subcutaneous (SC) doses of 1 and 15 mg/kg in humans were projected to be the minimally and the fully pharmacologically active doses, respectively. Based on the minimal anticipated biological effect level (MABEL) approach for starting dose selection, the dose of 0.05 mg/kg (3 mg for a 60 kg human) SC was recommended as the starting dose for FIH trials, because at this dose level < 10% target coverage was projected at Cmax (and all other time points). This study illustrates a rational mechanistic approach for the selection of FIH dose range for a therapeutic protein with a complex model of action. PMID:23529133

(1S,2R,3S,4R,5S)-4-(2-((5-Chlorothiophen-2-yl)ethynyl)-6-(methylamino)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-1-carboxamide (MRS5980) is an A3AR selective agonist containing multiple receptor affinity- and selectivity-enhancing modifications and a therapeutic candidate drug for many inflammatory diseases. Metabolism-related poor pharmacokinetic behavior and toxicities are a major reason for drug R&D failure. Metabolomics with UPLC-MS was employed to profile the metabolism of MRS5980 and MRS5980-induced disruption of endogenous compounds. Recombinant drug-metabolizing enzymes screening experiment were used to determine the enzymes involved in MRS5980 metabolism. Analysis of lipid metabolism-related genes was performed to investigate the reason for MRS5980-induced lipid metabolic disorders. Unsupervised principal components analysis separated the control and MRS5980 treatment groups in feces, urine, and liver samples, but not in bile and serum. The major ions mainly contributing to the separation of feces and urine were oxidized MRS5980, glutathione (GSH) conjugates and cysteine conjugate (degradation product of the GSH conjugates) of MRS5980. The major ions contributing to the group separation of liver samples were phosphatidylcholines. In vitro incubation experiments showed the involvement of CYP3A enzymes in the oxidative metabolism of MRS5980 and direct GSH reactivity of MRS5980. The electrophilic attack by MRS5980 is a minor pathway and did not alter GSH levels in liver or liver histology, and thus may be of minor clinical consequence. Gene expression analysis further showed decreased expression of PC biosynthetic genes choline kinase a and b, which further accelerated conversion of lysophosphatidylcholine to phosphatidylcholines through increasing the expression of lysophosphatidylcholine acyltransferase 3. These data will be useful to guide rational design of drugs targeting A3AR, considering efficacy, metabolic elimination, and

In rainbow trout, the food intake inhibition induced by serotonin occurs through 5-HT2C and 5-HT1A receptors, though the mechanisms involved are still unknown. Therefore, we assessed if a direct stimulation of 5-HT2C and 5-HT1A serotonin receptors (resulting in decreased food intake in rainbow trout), affects gene expression of neuropeptides involved in the control of food intake, such as pro-opiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART), corticotrophin releasing factor (CRF), and agouti-related peptide (AgRP). In a first set of experiments, the injection of the 5-HT2Creceptoragonists MK212 (60 μg kg(-1) icv) and WAY 161503 (1 mg kg(-1) ip), and of the 5-HT1A receptoragonist 8-OH-DPAT (1 mg kg(-1) ip and 30 μg kg(-1) icv) induced food intake inhibition. In a second set of experiments, we observed that the injection of MK212 or WAY 161503 (1 and 3 mg kg(-1)) significantly increased hypothalamic POMC mRNA abundance. CART mRNA abundance in hypothalamus was enhanced by treatment with MK212 and unaffected by WAY 161503. The administration of the 5-HT1A receptoragonist 8-OH-DPAT did not induce any significant variation in the hypothalamic POMC or CART mRNA levels. CRF mRNA abundance was only affected by MK212 that increased hypothalamic values. Finally, hypothalamic AgRP mRNA abundance was only evaluated with the agonist 5-HT2C MK212 resulting in no significant effects. The results show that the reduction in food intake mediated by 5-HT2Creceptors is associated with increases in hypothalamic POMC, CART and CRF mRNA abundance. PMID:26832922

Agomelatine (AGM), an analog of melatonin, is a potential agonist at melatonin receptors 1/2 and a selective antagonist at 5-hydroxytryptamine 2Creceptors. AGM is widely used for the treatment of major depressive episodes in adults. However, multiple adverse effects associated with AGM have been reported in clinical practice. It is little known about AGM metabolism in vitro and in vivo, although metabolism plays a pivotal role in its efficacy and safety. To elucidate metabolic pathways of AGM, we systemically investigated AGM metabolism and its bioactivation in human liver microsomes (HLM) and mice using metabolomic approaches. We identified thirty-eight AGM metabolites and adducts, among which thirty-two are novel. In HLM, we uncovered five GSH-trapped adducts and two semicarbazide-trapped aldehydes. Moreover, we characterized three N-acetyl cysteine conjugated-AGM adducts in mouse urine and feces, which were formed from the degradation of AGM_GSH adducts. Using recombinant CYP450 isoenzymes and chemical inhibitors, we demonstrated that CYP1A2 and CYP3A4 are primary enzymes contributing to the formation of AGM_GSH adducts and AGM_hydrazones. This study provided a global view of AGM metabolism and identified the novel pathways of AGM bioactivation, which could be utilized for further understanding the mechanism of adverse effects related to AGM and possible drug-drug interactions. PMID:27021842

We describe a new class of subunit-selective antagonists of N-methyl D-Aspartate (NMDA)-selective ionotropic glutamate receptors that contain the (E)-3-phenyl-2-styrylquinazolin-4(3H)-one backbone. The inhibition of recombinant NMDA receptor function induced by these quinazolin-4-one derivatives is non-competitive and voltage-independent, suggesting that this family of compounds does not exert action on the agonist binding site of the receptor or block the channel pore. The compounds described here resemble CP-465,022 ((S)-3-(2-chlorophenyl)-2-[2-(6-diethylaminomethyl-pyridin-2-yl)-vinyl]-6-fluoro-3H-quinazolin-4-one), a non-competitive antagonist of AMPA-selective glutamate receptors. However, modification of ring substituents resulted in analogues with greater than 100-fold selectivity for recombinant NMDA receptors over AMPA and kainate receptors. Furthermore, within this series of compounds, analogues were identified with 50-fold selectivity for recombinant NR2C/D-containing receptors over NR2A/B containing receptors. These compounds represent a new class of non-competitive subunit-selective NMDA receptor antagonists. PMID:20684595

Objective Ghrelin is known to regulate appetite control and cellular metabolism. The Corticotropin-Releasing Factor (CRF) family is also known to regulate energy balance. In this study, we investigated the links between ghrelin and the CRF family in C2C12 cells, a mouse myoblast cell line. Design and methods C2C12 cells were treated with ghrelin in the presence or absence of CRF receptor antagonists and then subjected to different metabolic analyses. Results Ghrelin enhanced glucose uptake by C2C12 cells, induced GLUT4 translocation to the cell surface and decreased RBP4 expression. A CRF-R2 selective antagonist, anti-sauvagine-30, blocked ghrelin-induced glucose uptake, Ghrelin upregulated CRF-R2 but not CRF-R1 levels. Moreover, ghrelin-treated C2C12 cells displayed a cAMP and pERK activation in response to Ucn3, a CRF-R2 specific ligand, but not in response to CRF or stressin, CRF-R1 specific ligands. Ghrelin also induced UCP2 and UCP3 expression, which were blocked by anti-sauvagine-30. Ghrelin did not induce fatty acids uptake by C2C12 cells or ACC expression. Even though C2C12 cells clearly exhibited responses to ghrelin, the known ghrelin receptor, GHSR1a, was not detectable in C2C12 cells. Conclusion Our results suggest that, ghrelin plays a role in regulating muscle glucose and, raise the possibility that suppression of the CRF-R2 pathway might provide benefits in high ghrelin states. PMID:23804489

SB 242084 has a high affinity (pKi 9.0) for the cloned human 5-HT2Creceptor and 100- and 158-fold selectivity over the closely related cloned human 5-HT2B and 5-HT2A subtypes respectively. SB 242084 had over 100-fold selectivity over a range of other 5-HT, dopamine and adrenergic receptors. In studies of 5-HT-stimulated phosphatidylinositol hydrolysis using SH-SY5Y cells stably expressing the cloned human 5-HT2Creceptor, SB 242084 acted as an antagonist with a pKb of 9.3, which closely resembled its corresponding receptor binding affinity. SB 242084 potently inhibited m-chlorophenylpiperazine (mCPP, 7 mgkg i.p. 20 min pre-test)-induced hypolocomotion in rats, a model of in vivo central 5-HT2Creceptor function, with an ID50 of 0.11 mg/kg i.p., and 2.0 mg/kg p.o. SB 242084 (0.1-1 mg/kg i.p.) exhibited an anxiolytic-like profile in the rat social interaction test, increasing time spent in social interaction, but having no effect on locomotion. SB 242084 (0.1-1 mg/kg i.p.) also markedly increased punished responding in a rat Geller-Seifter conflict test of anxiety, but had no consistent effect on unpunished responding. A large acute dose of SB 242084 (30 mg/kg p.o.) had no effect on seizure susceptibility in the rat maximal electroshock seizure threshold test. Also, while SB 242084 (2 and 6 mg/kg p.o. 1 hr pre-test) antagonized the hypophagic response to mCPP, neither acute nor subchronic administration of the drug, for 5 days at 2 or 6 mg/kg p.o. twice daily, affected food intake or weight gain. The results suggest that SB 242084 is the first reported selective potent and brain penetrant 5-HT2Creceptor antagonist and has anxiolytic-like activity, but does not possess either proconvulsant or hyperphagic properties which are characteristic of mutant mice lacking the 5-HT2Creceptor. PMID:9225286

The effects of ascorbic acid on dopaminergic /sup 3/H-agonistreceptor binding were studied in membrane homogenates of bovine anterior pituitary and caudate, and rat striatum. In all tissues virtually no stereospecific binding (defined using 1uM (+)butaclamol) of the /sup 3/H-agonists N-propylnorapomorphine (NPA), apomorphine, or dopamine could be demonstrated in the absence of ascorbic acid. Although levels of total /sup 3/H-agonist binding were three to five times greater in the absence than in the presence of 0.1% ascorbic acid, the increased binding was entirely non-stereospecific. Greater amounts of dopamine-inhibitable /sup 3/H-NPA binding could be demonstrated in the absence of 0.1% ascorbic acid, but this measure of ''specific binding'' was demonstrated not to represent dopamine receptor binding since several other catecholamines and catechol were equipotent with dopamine and more potent than the dopamine agonist (+/-)amino-6,7-dihydroxy-1,2,3,4-tetrahydronapthalene (ADTN) in inhibiting this binding. High levels of dopamine-displaceable /sup 3/H-agonist binding were detected in fresh and boiled homogenates of cerebellum, an area of brain which receives no dopaminergic innervation, further demonstrating the non-specific nature of /sup 3/H-agonist binding in the absence of ascorbic acid. These studies emphasize that under typical assay conditions ascorbic acid is required in order to demonstrate reversible and specific /sup 3/H-agonist binding to dopamine receptors.

Obective Glucagon-like peptide-1 (GLP-1) receptoragonists are indicated for treatment of type 2 diabetes since they mimic the actions of native GLP-1 on pancreatic islet cells, stimulating insulin release, while inhibiting glucagon release, in a glucose-dependent manner. The observation of weight loss has led to exploration of their potential as antiobesity agents, with liraglutide 3.0 mg day−1 approved for weight management in the US on December 23, 2014, and in the EU on March 23, 2015. This review examines the potential nonglycemic effects of GLP-1 receptoragonists. Methods A literature search was conducted to identify preclinical and clinical evidence on nonglycemic effects of GLP-1 receptoragonists. Results GLP-1 receptors are distributed widely in a number of tissues in humans, and their effects are not limited to the well-recognized effects on glycemia. Nonglycemic effects include weight loss, which is perhaps the most widely recognized nonglycemic effect. In addition, effects on the cardiovascular, neurologic, and renal systems and on taste perception may occur independently of weight loss. Conclusions GLP-1 receptoragonists may provide other nonglycemic clinical effects besides weight loss. Understanding these effects is important for prescribers in using GLP-1 receptoragonists for diabetic patients, but also if approved for chronic weight management. PMID:25959380

The roles of lateral habenular nucleus (LHb) glutamate neurons and serotonin2C (5-HT2C) receptors in depression are poorly understood, particularly in Parkinson's disease-associated depression. Here we assessed the importance of LHb glutamate neurons and 5-HT2Creceptors for depressive-like behaviors in sham-operated rats and rats with unilateral 6-hydroxydopamine lesions of the substantia nigra. The lesion induced depressive-like responses compared to sham-operated rats. Intra-LHb injection of potent, selective 5-HT2Creceptoragonist Ro60-0175 decreased sucrose consumption and increased immobility time in sham-operated rats, indicating the induction of depressive-like responses, and intra-LHb injection of Ro60-0175 further increased the expression of depressive-like behaviors in the lesioned rats. Activation of LHb 5-HT2Creceptors by the local administration of Ro60-0175 increased the firing rate of EAAC1 (a neuronal glutamate transporter)-positive neurons and percentage of the neurons with burst-firing pattern in the two groups of rats. Compared to sham-operated rats, the duration of Ro60-0175 action on the firing rate of EAAC1-positive neurons was markedly prolonged in the lesioned rats. Intra-LHb injection of Ro60-0175 decreased dopamine, 5-HT and noradrenaline levels in the medial prefrontal cortex, habenula, hippocampus and amygdala in sham-operated and the lesioned rats. The lesion did not change the percentage of EAAC1/5-HT2Creceptor co-expressing neurons in the LHb. These findings indicate that activation of 5-HT2Creceptors in the LHb increases firing activity of LHb glutamate neurons and then decreases monoamine levels in several brain regions, which increase the expression of depressive-like behaviors. Further, our results also suggest that the lesion leads to hyperfunctionality of 5-HT2Creceptors on glutamate neurons of the LHb. PMID:25661701

The repeated administration of selective kappa-opioid receptoragonists prevents the locomotor activation produced by acute cocaine administration and the development of cocaine-induced behavioral sensitization. Previous studies have shown that dopamine (DA) D2 autoreceptors modulate the synthesis and release of DA in the striatum. Evidence that kappa agonist treatment downregulates DA D2 receptors in this same brain region has recently been obtained. Accordingly, the present studies were undertaken to examine the influence of repeated kappa-opioid receptoragonist administration on pre- and postsynaptic DA D2 receptor function in the dorsal striatum using pre- and postsynaptic receptor-selective doses of quinpirole. Rats were injected once daily with the selective kappa-opioid receptoragonist U69593 (0.16-0.32 mg/kg s.c.) or vehicle for 3 days. Microdialysis studies assessing basal and quinpirole-evoked (0.05 mg/kg s.c.) DA levels were conducted 2 days later. Basal and quinpirole-stimulated locomotor activity were assessed in a parallel group of animals. The no-net flux method of quantitative microdialysis revealed no effect of U69593 on basal DA dynamics, in that extracellular DA concentration and extraction fraction did not differ in control and U69593-treated animals. Acute administration of quinpirole significantly decreased striatal DA levels in control animals, but in animals treated with U69593, the inhibitory effects of quinpirole were significantly reduced. Quinpirole produced a dose-related increase in locomotor activity in control animals, and this effect was significantly attenuated in U69593-treated animals. These data reveal that prior repeated administration of a selective kappa-opioid receptoragonist attenuates quinpirole-induced alterations in DA neurotransmission and locomotor activity. These results suggest that both pre- and postsynaptic striatal DA D2 receptors may be downregulated following repeated kappa-opioid receptoragonist

The development of small molecule agonists for class B G protein-coupled receptors (GPCRs) has been quite challenging. With proof-of-concept that exenatide, the parenterally administered peptide agonist of the glucagon-like peptide-1 (GLP1) receptor, is an effective treatment for patients with diabetes mellitus, the development of small molecule agonists could have substantial advantages. We previously reported a lead for small molecule GLP1 receptoragonist development representing the pentapeptide NRTFD. In this work, we have prepared an NRTFD derivative incorporating a photolabile benzoylphenylalanine and used it to define its site of action. This peptide probe was a full agonist with potency similar to NRTFD, which bound specifically and saturably to a single, distinct site within the GLP1 receptor. Peptide mapping using cyanogen bromide and endoproteinase Lys-C cleavage of labeled wild type and M397L mutant receptor constructs identified the site of covalent attachment of NRTFD within the third extracellular loop above the sixth transmembrane segment. This region is the same as that identified using an analogous photolabile probe based on secretin receptor sequences, and has been shown in mutagenesis studies to be important for natural agonist action of several members of this family. While these observations suggest that small molecule ligands can act at a site bordering the third extracellular loop to activate this class B GPCR, the relationship of this site to the site of action of the amino-terminal end of the natural agonist peptide is unclear. PMID:22079758

We established a transgenic silkworm strain expressing the human insulin receptor (hIR) using the GAL4/UAS system. Administration of human insulin to transgenic silkworms expressing hIR decreased hemolymph sugar levels and facilitated Akt phosphorylation in the fat body. The decrease in hemolymph sugar levels induced by injection of human insulin in the transgenic silkworms expressing hIR was blocked by co-injection of wortmannin, a phosphoinositide 3-kinase inhibitor. Administration of bovine insulin, an hIR ligand, also effectively decreased sugar levels in the transgenic silkworms. These findings indicate that functional hIRs that respond to human insulin were successfully induced in the transgenic silkworms. We propose that the humanized silkworm expressing hIR is useful for in vivo evaluation of the therapeutic activities of insulin receptoragonists. PMID:25449269

1. The 5-HT2 receptoragonist activity of fenoldopam (SKF 82526) was characterized in the rabbit isolated aorta preparation. 2. Fenoldopam was an agonist at the vascular 5-HT2 receptor with lower affinity and efficacy than the naturally occurring agonist 5-hydroxytryptamine (5-HT). Fenoldopam had an affinity (pKA) of 5.84 +/- 0.04 and efficacy (tau) of 0.57 +/- 0.04, whereas 5-HT had a pKA of 6.65 +/- 0.12 and tau of 2.66 +/- 0.41. 3. The constrictor effects of fenoldopam and 5-HT were competitively antagonized by the 5-HT2 antagonist, ketanserin, with pKB values of 8.81 +/- 0.11 and 8.83 +/- 0.10 respectively. 4. Prior incubation with fenoldopam produced a concentration-related rightward shift of a subsequent 5-HT concentration-response curve. This inhibition was specific for 5-HT since constrictor responses to angiotensin II were unaffected. 5. This study indicates that the D1 receptoragonist, fenoldopam, acts as an agonist at the vascular 5-HT2 receptor, but with an affinity and efficacy less than that of the naturally occurring agonist, 5-HT. PMID:1361397

Toll-like receptors (TLR) are members of a highly conserved group of receptors which recognize conserved molecular aspects of microbes. The purpose of these experiments were to ascertain the effects of the administration of the TLR 9 agonist, CpG, on the colonization of neonatal swine with Salmonel...

The identification of sigma receptor (σR) subtypes has been based on radioligand binding and, despite progress with σ1R cellular function, less is known about σR subtype functions in vivo. Recent findings that cocaine self administration experience will trigger σR agonist self administration was used in this study to assess the in vivo receptor subtype specificity of the agonists (+)-pentazocine, PRE-084 [2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate hydrochloride], and 1,3-di-o-tolylguanidine (DTG) and several novel putative σR antagonists. Radioligand binding studies determined in vitro σR selectivity of the novel compounds, which were subsequently studied for self administration and antagonism of cocaine, (+)-pentazocine, PRE-084, or DTG self administration. Across the dose ranges studied, none of the novel compounds were self administered, nor did they alter cocaine self administration. All compounds blocked DTG self administration, with a subset also blocking (+)-pentazocine and PRE-084 self administration. The most selective of the compounds in binding σ1Rs blocked cocaine self administration when combined with a dopamine transport inhibitor, either methylphenidate or nomifensine. These drug combinations did not decrease rates of responding maintained by food reinforcement. In contrast, the most selective of the compounds in binding σ2Rs had no effect on cocaine self administration in combination with either dopamine transport inhibitor. Thus, these results identify subtype-specific in vivo antagonists, and the utility of σR agonist substitution for cocaine self administration as an assay capable of distinguishing σR subtype selectivity in vivo. These results further suggest that effectiveness of dual σR antagonism and dopamine transport inhibition in blocking cocaine self administration is specific for σ1Rs and further support this dual targeting approach to development of cocaine antagonists. PMID:27189970

The identification of sigma receptor (σR) subtypes has been based on radioligand binding and, despite progress with σ1R cellular function, less is known about σR subtype functions in vivo. Recent findings that cocaine self administration experience will trigger σR agonist self administration was used in this study to assess the in vivo receptor subtype specificity of the agonists (+)-pentazocine, PRE-084 [2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate hydrochloride], and 1,3-di-o-tolylguanidine (DTG) and several novel putative σR antagonists. Radioligand binding studies determined in vitro σR selectivity of the novel compounds, which were subsequently studied for self administration and antagonism of cocaine, (+)-pentazocine, PRE-084, or DTG self administration. Across the dose ranges studied, none of the novel compounds were self administered, nor did they alter cocaine self administration. All compounds blocked DTG self administration, with a subset also blocking (+)-pentazocine and PRE-084 self administration. The most selective of the compounds in binding σ1Rs blocked cocaine self administration when combined with a dopamine transport inhibitor, either methylphenidate or nomifensine. These drug combinations did not decrease rates of responding maintained by food reinforcement. In contrast, the most selective of the compounds in binding σ2Rs had no effect on cocaine self administration in combination with either dopamine transport inhibitor. Thus, these results identify subtype-specific in vivo antagonists, and the utility of σR agonist substitution for cocaine self administration as an assay capable of distinguishing σR subtype selectivity in vivo. These results further suggest that effectiveness of dual σR antagonism and dopamine transport inhibition in blocking cocaine self administration is specific for σ1Rs and further support this dual targeting approach to development of cocaine antagonists. PMID:27189970

γ-Hydroxybutyric acid (GHB) is an endogenous compound and a drug used clinically to treat the symptoms of narcolepsy. GHB is known to be an agonist of GABAB receptors with millimolar affinity, but also binds with much higher affinity to another site, known as the GHB receptor. While a body of evidence has shown that GHB does not bind to GABAA receptors widely, recent evidence has suggested that the GHB receptor is in fact on extrasynaptic α4β1δ GABAA receptors, where GHB acts as an agonist with an EC50 of 140 nM. We investigated three neuronal cell types that express a tonic GABAA receptor current mediated by extrasynaptic receptors: ventrobasal (VB) thalamic neurons, dentate gyrus granule cells and striatal medium spiny neurons. Using whole-cell voltage clamp in brain slices, we found no evidence that GHB (10 µM) induced any GABAA receptor mediated current in these cell types, nor that it modulated inhibitory synaptic currents. Furthermore, a high concentration of GHB (3 mM) was able to produce a GABAB receptor mediated current, but did not induce any other currents. These results suggest either that GHB is not a high affinity agonist at native α4β1δ receptors, or that these receptors do not exist in classical areas associated with extrasynaptic currents. PMID:24244421

γ-Hydroxybutyric acid (GHB) is an endogenous compound and a drug used clinically to treat the symptoms of narcolepsy. GHB is known to be an agonist of GABAB receptors with millimolar affinity, but also binds with much higher affinity to another site, known as the GHB receptor. While a body of evidence has shown that GHB does not bind to GABAA receptors widely, recent evidence has suggested that the GHB receptor is in fact on extrasynaptic α4β1δ GABAA receptors, where GHB acts as an agonist with an EC50 of 140 nM. We investigated three neuronal cell types that express a tonic GABAA receptor current mediated by extrasynaptic receptors: ventrobasal (VB) thalamic neurons, dentate gyrus granule cells and striatal medium spiny neurons. Using whole-cell voltage clamp in brain slices, we found no evidence that GHB (10 µM) induced any GABAA receptor mediated current in these cell types, nor that it modulated inhibitory synaptic currents. Furthermore, a high concentration of GHB (3 mM) was able to produce a GABAB receptor mediated current, but did not induce any other currents. These results suggest either that GHB is not a high affinity agonist at native α4β1δ receptors, or that these receptors do not exist in classical areas associated with extrasynaptic currents. PMID:24244421

Aiming to discover dual-acting β2 adrenergic/dopamine D2 receptor ligands, a structure-guided approach for the evolution of GPCR agonists that address multiple targets was elaborated. Starting from GPCR crystal structures, we describe the design, synthesis and biological investigation of a defined set of compounds leading to the identification of the benzoxazinone (R)-3, which shows agonist properties at the adrenergic β2 receptor and substantial G protein-promoted activation at the D2 receptor. This directed approach yielded molecular probes with tuned dual activity. The congener desOH-3 devoid of the benzylic hydroxyl function was shown to be a β2 adrenergic antagonist/D2 receptoragonist with Ki values in the low nanomolar range. The compounds may serve as a promising starting point for the investigation and treatment of neurological disorders. PMID:27132867

The D-1 dopamine receptor was extracted from rat striatal membranes with sodium cholate and NaCl in the presence of a specific agonist and phospholipids. The soluble receptor then was reconstituted into phospholipid vesicles by further addition of phospholipids prior to detergent removal. Of the total membrane receptors, up to 48% were extracted and 36% were reconstituted into phospholipid vesicles. Yields were greatly reduced if the agonist was omitted or replaced with an antagonist. The solubilized and reconstituted D-1 receptors retained the pharmacological properties of the membrane-bound receptors, including the ability to discriminate between active and inactive enantiomers of specific agonists and antagonists. In this regard, the affinity of the reconstituted receptors for the D-1 specific antagonist /sup 125/I SCH 23982 was similar to that of the membrane-bound receptors with a Kd of 1.5 nM. Both the soluble and reconstituted forms of the D-1 receptor exhibited two affinity states for the D-1 specific agonist SKandF R-38393. In contrast to the low proportion of the receptors that had a high affinity for the agonists in striatal membranes (less than 6%), there was a dramatic increase following solubilization (22%) and reconstitution (40%). Similar results were obtained by using dopamine; the proportion of high-affinity sites increased from 4% (membrane-bound) to 48% (reconstituted) of the total receptor population. These high-affinity sites were coupled to G proteins, as guanyl nucleotides completely abolished them. Addition of guanyl nucleotides prior to solubilization or to reconstitution, however, had no effect on the subsequent yield of the reconstituted receptors.

The adrenergic system has been hypothesized to be involved in the etiology of attention-deficit hyperactivity disorder (ADHD) based on pharmacological interventions and animal models. Noradrenergic neurons are implicated in the modulation of vigilance, improvement of visual attention, initiation of adaptive response, learning and memory. In this study we tested the genes for two adrenergic receptors, alpha 1C (ADRA1C) located on chromosome 8p11.2, and alpha 2C (ADRA2C) located on chromosome 4p16, as genetic susceptibility factors in ADHD. For the adrenergic receptor alpha 1C we used a C to T polymorphism that results in a change of Cys to Arg at codon 492 for the linkage study. For the adrenergic receptor alpha 2C gene we examined a dinucleotide repeat polymorphism located approximately 6 kb from the gene. We examined these polymorphisms in a sample of 103 families ascertained through an ADHD proband. Using the transmission disequilibrium test, we did not observe biased transmission of any of the alleles of these polymorphisms. We conclude that the alleles at the polymorphisms tested in these two genes are not linked to the ADHD phenotype in this sample of families. PMID:11326305

Both the 5-HT(2A) receptor (R) antagonist M100907 and the 5-HT(2C) R agonist MK212 attenuate cocaine-induced dopamine release and hyperlocomotion. This study examined whether these drugs interact to reduce cocaine hyperlocomotion and Fos expression in the striatum and prefrontal cortex. We first determined from dose-effect functions a low dose of both M100907 and MK212 that failed to alter cocaine (15 mg/kg, i.p.) hyperlocomotion. Subsequently, we examined whether these subthreshold doses given together would attenuate cocaine hyperlocomotion, consistent with a 5-HT(2A)/5-HT(2C) R interaction. Separate groups of rats received two sequential drug injections 5 min apart immediately before a 1-h locomotion test as follows: (1) saline + saline, (2) saline + cocaine, (3) 0.025 mg/kg M100907 + cocaine, (4) 0.125 mg/kg MK212 + cocaine, or (5) cocktail combination of 0.025 mg/kg M100907 and 0.125 mg/kg MK212 + cocaine. Brains were extracted for Fos immunohistochemistry 90 min after the second injection. We next examined the effects of 0.025 mg/kg M100907 and 0.125 mg/kg MK212, alone and in combination, on spontaneous locomotor activity. While neither drug given alone produced any effects, the M100907/MK212 cocktail attenuated cocaine hyperlocomotion as well as cocaine-induced Fos expression in the dorsolateral caudate-putamen (CPu), but had no effect on spontaneous locomotion. The findings suggest that 5-HT(2A) Rs and 5-HT(2C) Rs interact to attenuate cocaine hyperlocomotion and Fos expression in the CPu, and that the CPu is a potential locus of the interactive effects between these 5-HT(2) R subtypes on behavior. Further research investigating combined 5-HT(2A) R antagonism and 5-HT(2C) R agonism as a treatment for cocaine dependence is warranted. PMID:22886755

Lepidozenolide is a sesquiterpenoid isolated from the liverwort Lepidozia fauriana and its possible bioactivity is unclear. The farnesoid X receptor (FXR) is a member of nuclear receptor superfamily that has been widely targeted for developing treatments for chronic liver disease and hyperglycemia. In this study, whether lepidozenolide may act as a FXR agonist was determined. Indeed, in mammalian one-hybrid and transient transfection reporter assays, lepidozenolide transactivated FXR to modulate promoter action including GAL4, CYP7A1, and PLTP promoters in a dose-dependent manner, while it exhibited slightly less agonistic activity than chenodeoxycholic acid, an endogenous FXR agonist. Through the molecular modeling docking studies lepidozenolide was shown to bind to FXR ligand binding pocket fairly well. All these results indicate that lepidozenolide acts as a FXR agonist. PMID:25315435

This review examines the growing literature on the role of peroxisome proliferator-activated receptors (PPARs) in addiction. There are two subtypes of PPAR receptors that have been studied in addiction: PPAR-α and PPAR-γ. The role of each PPAR subtype in common models of addictive behavior, mainly pre-clinical models, is summarized. In particular, studies are reviewed that investigated the effects of PPAR-α agonists on relapse, sensitization, conditioned place preference, withdrawal and drug intake, and effects of PPAR-γ agonists on relapse, withdrawal and drug intake. Finally, studies that investigated the effects of PPAR agonists on neural pathways of addiction are reviewed. Taken together this preclinical data indicates that PPAR agonists are promising new medications for drug addiction treatment. PMID:23614675

The imidazoline I2 receptor is an emerging drug target for analgesics. This study extended previous studies by examining the antinociceptive effects of three I2 receptoragonists (2-BFI, BU224, and CR4056) in the formalin test. The receptor mechanisms and anatomical mediation of I2 receptoragonist-induced antinociception were also examined. Formalin-induced flinching responses (2%, 50 μl) were quantified after treatment with I2 receptoragonists alone or in combination with the I2 receptor antagonist idazoxan. Anatomical mediation was studied by locally administering 2-BFI into the plantar surface or into the right lateral ventricle through cannulae (intracerebroventricular). The locomotor activity was also examined after central (intracerebroventricular) administration of 2-BFI. 2-BFI (1-10 mg/kg, intraperitoneal) and BU224 (1-10 mg/kg, intraperitoneal) attenuated the spontaneous flinching response observed during 10 min (phase 1) and 20-60 min (phase 2) following formalin treatment, whereas CR4056 (1-32 mg/kg, intraperitoneal) decreased only phase 2 flinching response. The I2 receptor antagonist idazoxan attenuated the antinociceptive effects of 2-BFI and BU224 during phase 1, but not phase 2. Peripheral administration of 2-BFI (1-10 mg/kg, intraplantar) to the hind paw of rats had no antinociceptive effect. In contrast, centrally delivered 2-BFI (10-100 µg, intracerebroventricular) dose-dependently attenuated phase 1 and phase 2 flinching at doses that did not reduce the locomotor activity. Together, these data revealed the differential antinociceptive effects of I2 receptoragonists and the differential antagonism profiles by idazoxan, suggesting the involvement of different I2 receptor subtypes in reducing different phases of formalin-induced pain-like behaviors. In addition, the results also suggest the central mediation of I2 receptoragonist-induced antinociceptive actions. PMID:26599907

Clinical studies have shown that agonist-antagonist opioid analgesics that produce their analgesic effect via action on the kappa-opioid receptor, produce a delayed-onset anti-analgesia in men but not women, an effect blocked by co-administration of a low dose of naloxone. We now report the same time-dependent anti-analgesia and its underlying mechanism in an animal model. Using the Randall-Selitto paw-withdrawal assay in male rats, we found that nalbuphine, pentazocine, and butorphanol each produced analgesia during the first hour followed by anti-analgesia starting at ∼90min after administration in males but not females, closely mimicking its clinical effects. As observed in humans, co-administration of nalbuphine with naloxone in a dose ratio of 12.5:1 blocked anti-analgesia but not analgesia. Administration of the highly selective kappa-opioid receptoragonist U69593 produced analgesia without subsequent anti-analgesia, and confirmed by the failure of the selective kappa antagonist nor-binaltorphimine to block nalbuphine-induced anti-analgesia, indicating that anti-analgesia is not mediated by kappa-opioid receptors. We therefore tested the role of other receptors in nalbuphine anti-analgesia. Nociceptin/orphanin FQ (NOP) and sigma-1 and sigma-2 receptors were chosen on the basis of their known anti-analgesic effects and receptor binding studies. The selective NOP receptor antagonists, JTC801, and J-113397, but not the sigma receptor antagonist, BD 1047, antagonized nalbuphine anti-analgesia. Furthermore, the NOP receptoragonist NNC 63-0532 produced anti-analgesia with the same delay in onset observed with the three agonist-antagonists, but without producing preceding analgesia and this anti-analgesia was also blocked by naloxone. These results strongly support the suggestion that clinically used agonist-antagonists act at the NOP receptor to produce anti-analgesia. PMID:24188792

The amino acids glycine, β-alanine and taurine are all endogenous agonists of the glycine receptor. In this study, a combination of rapid agonist application onto macropatches and steady-state single-channel recordings was used to compare the actions of glycine, β-alanine and taurine upon homomeric α1 human glycine receptors transiently expressed in human embryonic kidney (HEK 293) cells. The 10–90 % rise times determined from rapid application of 100 μm of each agonist were indistinguishable, indicating each agonist has a similar association rate. At saturating concentrations (30 mm) the rise time for glycine (0.26 ms) was 1.8-fold faster than that for β-alanine (0.47 ms) and 3.9-fold faster than that for taurine (1.01 ms), indicating clear differences in the maximum opening rate between agonists. The relaxation following rapid removal of agonist was fitted with a single exponential for β-alanine (3.0 ms) and taurine (2.2 ms), and two exponential components for glycine with a weighted mean time constant of 27.1 ms. This was consistent with differences in dissociation rates estimated from analysis of bursts, with taurine > β-alanine > glycine. Exponential fits to the open period distributions gave time constants that did not differ between agonists and the geometric distribution for the number of openings per burst indicated that all three agonists had a significant component of single-opening bursts. Based upon these data, we propose a kinetic scheme with three independent open states, where the opening rates are dependent upon the activating agonist, while the closing rates are an intrinsic characteristic of the receptor. PMID:12679369

The compound 4-(5-(4-bromophenyl)-3-(6-methyl-2-oxo-4-phenyl-1,2-dihydroquinolin-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)-4-oxobutanoic acid (DQP-1105) is a representative member of a new class of N-methyl-d-aspartate (NMDA) receptor antagonists. DQP-1105 inhibited GluN2C- and GluN2D-containing receptors with IC50 values that were at least 50-fold lower than those for recombinant GluN2A-, GluN2B-, GluA1-, or GluK2-containing receptors. Inhibition was voltage-independent and could not be surmounted by increasing concentrations of either coagonist, glutamate or glycine, consistent with a noncompetitive mechanism of action. DQP-1105 inhibited single-channel currents in excised outside-out patches without significantly changing mean open time or single-channel conductance, suggesting that DQP inhibits a pregating step without changing the stability of the open pore conformation and thus channel closing rate. Evaluation of DQP-1105 inhibition of chimeric NMDA receptors identified two key residues in the lower lobe of the GluN2 agonist binding domain that control the selectivity of DQP-1105. These data suggest a mechanism for this new class of inhibitors and demonstrate that ligands can access, in a subunit-selective manner, a new site located in the lower, membrane-proximal portion of the agonist-binding domain. PMID:21807990

Chronic pain negatively impacts the quality of life in a variety of patient populations. The current therapeutic repertoire is inadequate in managing patient pain and warrants the development of new therapeutics. Adenosine and its four cognate receptors (A1 , A2A , A2B and A3 ) have important roles in physiological and pathophysiological states, including chronic pain. Preclinical and clinical studies have revealed that while adenosine and agonists of the A1 and A2A receptors have antinociceptive properties, their therapeutic utility is limited by adverse cardiovascular side effects. In contrast, our understanding of the A3 receptor is only in its infancy, but exciting preclinical observations of A3 receptor antinociception, which have been bolstered by clinical trials of A3 receptoragonists in other disease states, suggest pain relief without cardiovascular side effects and with sufficient tolerability. Our goal herein is to briefly discuss adenosine and its receptors in the context of pathological pain and to consider the current data regarding A3 receptor-mediated antinociception. We will highlight recent findings regarding the impact of the A3 receptor on pain pathways and examine the current state of selective A3 receptoragonists used for these studies. The adenosine-to-A3 receptor pathway represents an important endogenous system that can be targeted to provide safe, effective pain relief from chronic pain. PMID:26804983

G protein-coupled receptors, such as the adenosine A2A receptor, are dynamic proteins, which undergo agonist-dependent redistribution from the cell surface to intracellular membranous compartments, such as endosomes. In order to study the kinetics of adenosine A2A receptor redistribution in living cells, we synthesized a novel fluorescent agonist, Alexa488-APEC. Alexa488-APEC binds to adenosine A2A (Ki = 149 ± 27 nM) as well as A3 receptors (Ki= 240 ± 160 nM) but not to adenosine A1 receptors. Further, we characterized the dose-dependent increase in Alexa488-APEC-induced cAMP production as well as cAMP response element binding (CREB) protein phosphorylation, verifying the ligand’s functionality at adenosine A2A but not A2B receptors. In live cell imaging studies, Alexa488-APEC induced adenosine A2A receptor internalization, which was blocked by the competitive reversible antagonist ZM 241385 and hyperosmolaric sucrose. Further, internalized adenosine A2A receptors co-localized with clathrin and Rab5, indicating that agonist stimulation promotes adenosine A2A receptor uptake through a clathrin-dependent mechanism to Rab5-positive endosomes. The basic characterization of Alexa488-APEC provided here showed that it provides a usefultool for tracing adenosine A2A receptors in vitro. PMID:18603240

To differentiate the opiate (naloxone) receptor and the enkephalin receptor in rat brain, we solubilized the receptor molecules by detergent and determined the molecular weights by gel filtration. The receptor preparation was bound to [3H] naloxone or [3H] Met5-enkephalin, and was solubilized by Triton X-100. On gel chromatography with a Sepharose 6B column, the agonist and the antagonist conformation of opioid receptors eluted as molecules with the molecular weights of 240,000, and 120,000 and with Stokes' radii of 5.5 nm and 4.3 nm, respectively. Further, it was also disclosed that Na+ was bound to the antagonist conformation of opioid receptors but not to the agonist conformation. PMID:6275320

A series of biaryl pyrazole and imidazole Liver X Receptor (LXR) partial agonists has been synthesized displaying LXRβ selectivity. The LXRβ selective partial agonist 18 was identified with potent induction of ATP binding transporters ABCA1 and ABCG1 in human whole blood (EC50=1.2μM, 55% efficacy). In mice 18 displayed peripheral induction of ABCA1 at 3 and 10mpk doses with no significant elevation of plasma or hepatic triglycerides at these doses, showing an improved profile compared to a full pan-agonist. PMID:25435151

The adenosine A(2A) receptor (A(2A)R) is a G-protein-coupled receptor that plays a key role in transmembrane signalling mediated by the agonist adenosine. The structure of A(2A)R was determined recently in an antagonist-bound conformation, which was facilitated by the T4 lysozyme fusion in cytoplasmic loop 3 and the considerable stabilisation conferred on the receptor by the bound inverse agonist ZM241385. Unfortunately, the natural agonist adenosine does not sufficiently stabilise the receptor for the formation of diffraction-quality crystals. As a first step towards determining the structure of A(2A)R bound to an agonist, the receptor was thermostabilised by systematic mutagenesis in the presence of the bound agonist [(3)H]5'-N-ethylcarboxamidoadenosine (NECA). Four thermostabilising mutations were identified that when combined to give mutant A(2A)R-GL26, conferred a greater than 200-fold decrease in its rate of unfolding compared to the wild-type receptor. Pharmacological analysis suggested that A(2A)R-GL26 is stabilised in an agonist-bound conformation because antagonists bind with up to 320-fold decreased affinity. None of the thermostabilising mutations are in the ZM241385 binding pocket, suggesting that the mutations affect ligand binding by altering the conformation of the receptor rather than through direct interactions with ligands. A(2A)R-GL26 shows considerable stability in short-chain detergents, which has allowed its purification and crystallisation. PMID:21501622

Highlights: •Alkyl-LPA specifically interacts with PPARγ. •Alkyl-LPA treatments induces lipid accumulation in C2C12 cells. •Alkyl-LPA enhanced glucose uptake in C2C12 cells. •Alkyl-LPA-treated C2C12 cells express increased amounts of GLUT4 mRNA. •Alkyl-LPA is a novel therapeutic agent that can be used for the treatment of obesity and diabetes. -- Abstract: Studies on the effects of lipids on skeletal muscle cells rarely examine the effects of lysophospholipids. Through our recent studies, we identified select forms of phospholipids, such as alkyl-LPA, as ligands for the intracellular receptor peroxisome proliferator-activated receptor gamma (PPARγ). PPARγ is a nuclear hormone receptor implicated in many human diseases, including diabetes and obesity. We previously showed that alkyl-LPA is a specific agonist of PPARγ. However, the mechanism by which the alkyl-LPA–PPARγ axis affects skeletal muscle cells is poorly defined. Our objective in the present study was to determine whether alkyl-LPA and PPARγ activation promotes glucose uptake in skeletal muscle cells. Our findings indicate that PPARγ1 mRNA is more abundant than PPARγ2 mRNA in C2C12 cells. We showed that alkyl-LPA (3 μM) significantly activated PPARγ and increased intracellular glucose levels in skeletal muscle cells. We also showed that incubation of C2C12 cells with alkyl-LPA led to lipid accumulation in the cells. These findings suggest that alkyl-LPA activates PPARγ and stimulates glucose uptake in the absence of insulin in C2C12 cells. This may contribute to the plasma glucose-lowering effect in the treatment of insulin resistance.

A series of mono- and dimethyoxy substituted tolazoline derivatives, known to be partial agonists at the alpha/sub 1/ receptor, were compared with the ..cap alpha../sub 1/ selective full agonist phenylephrine (PE) on isolated strips of rabbit aorta Agonist activity was evaluated in contraction, /sup 45/Ca influx, /sup 45/Ca efflux, and /sup 32/P-Phospholipid labelling studies. Maximum contractile responses for the 2-, 3-, and 3, 5- methoxy substituted tolazoline derivatives (10/sup -5/M) were 53.8, 67.6 and 99.7% of the PE (10/sup -5/M) response respectively. These same partial agonists caused a stimulation of /sup 45/Ca influx to the extent of 64, 86, and 95% of the PE response respectively. In /sup 45/Ca efflux studies, (a measure of the intracellular Ca/sup +2/ release) the tolazolines caused: 30%, 63%, and 78% of the PE stimulated level. /sup 32/P-Phosphatidic acid (PA) labelling was measured as an index of PI turnover after ..cap alpha../sub 1/ receptor stimulation. Compared to PE, the 2-, 3-, and 3,5- methoxy substituted tolazoline derivatives caused 22, 46, and 72% PA labelling. The above values are all in reasonable accord with the rank order or agonist activity shown in maximum contractile responses. The results of this investigation suggest that partial agonists stimulate ..cap alpha.. receptor coupling events at a level which is quantitatively comparable to their potencies in causing contraction of arterial smooth muscle.

Vasoactive intestinal peptide (VIP) mediates a broad range of biological responses by activating two related receptors, VIP receptor 1 and 2 (VIPR1 and VIPR2). Although the use of native VIP facilitates neuroprotection, clinical application of the hormone is limited due to VIP's rapid metabolism and inability to distinguish between VIPR1 and VIPR2 receptors. In addition, activation of both receptors by therapeutics may increase adverse secondary toxicities. Therefore, we developed metabolically stable and receptor-selective agonists for VIPR1 and VIPR2 to improve pharmacokinetic and pharmacodynamic therapeutic end points. Selective agonists were investigated for their abilities to protect mice against MPTP-induced neurodegeneration used to model Parkinson's disease (PD). Survival of tyrosine hydroxylase neurons in the substantia nigra was determined by stereological tests after MPTP intoxication in mice pretreated with either VIPR1 or VIPR2 agonist or after adoptive transfer of splenic cell populations from agonist-treated mice administered to MPTP-intoxicated animals. Treatment with VIPR2 agonist or splenocytes from agonist-treated mice resulted in increased neuronal sparing. Immunohistochemical tests showed that agonist-treated mice displayed reductions in microglial responses, with the most pronounced effects in VIPR2 agonist-treated, MPTP-intoxicated mice. In parallel studies, we observed reductions in proinflammatory cytokine release that included IL-17A, IL-6, and IFN-γ and increases in GM-CSF transcripts in CD4+ T cells recovered from VIPR2 agonist-treated animals. Moreover, a phenotypic shift of effector to regulatory T cells was observed. These results support the use of VIPR2-selective agonists as neuroprotective agents for PD treatment. SIGNIFICANCE STATEMENT Vasoactive intestinal peptide receptor 2 can elicit immune transformation in a model of Parkinson's disease (PD). Such immunomodulatory capabilities can lead to neuroprotection by attenuating

Previous studies have demonstrated that serotonin 5-HT2Creceptors in the dorsal periaqueductal gray (dPAG) mediate both anxiety and antinociception in mice submitted to the elevated plus maze. The present study examined the effects of intra-dPAG infusion of the serotonin 5-HT2Creceptoragonist (MK-212) in the defensive reactions and antinociception in mice with neurophatic pain confronted by a predator. Neuropathic pain was induced by chronic constriction injury (CCI) of the sciatic nerve, and predator confrontation was performed using the rat exposure test (RET). Our results demonstrated that both sham-operated and CCI mice exhibited intense defensive reactions when confronted by rats. However, rat-exposed CCI mice showed reduced pain reactivity in comparison to CCI mice exposed to a toy rat. Intra-dPAG infusion of MK-212 prior to predator exposure did not significantly alter defensive or antinociceptive responses. To our knowledge, our results represent the first evidence of RET-induced antinociception in mice. Moreover, the results of the present study suggest that 5-HT2Creceptor activation in the dPAG is not critically involved in the control of predator-evoked fearful or antinociceptive responses. PMID:27059332

G protein-coupled receptors (GPCRs) are an important class of drug targets. Quantitative analysis by global curve fitting of properly designed dose-dependent GPCR agonism and allosterism data permits the determination of all affinity and efficacy parameters based on a general operational model. We report here a quantitative and panoramic measure of receptoragonist and modulator equi-response and equi-occupancy selectivity calculated from these parameters. The selectivity values help to differentiate not only one agonist or modulator from another, but on-target from off-target receptor or functional pathway as well. Furthermore, in conjunction with target site free drug concentrations and endogenous agonist tones, the allosterism parameters and selectivity values may be used to predict in vivo efficacy and safety margins. PMID:27116909

Prostacyclin controls cardiovascular function via activation of the prostacyclin receptor. Decreased prostacyclin production occurs in several cardiovascular diseases. However, the clinical use of prostacyclin and its analogues is complicated by their chemical and metabolic instability. A medicinal chemistry program searched for novel nonprostanoid prostacyclin receptoragonists not subject to these limitations. A compound with a diphenylpyrazine structural core was synthesized. Metabolic stability and agonist potency were optimized through modification of the linear side chain. Compound 12b (MRE-269, ACT-333679) was identified as a potent and highly selective prostacyclin receptoragonist. Replacement of the terminal carboxyl group with an N-acylsulfonamide group yielded parent compound 26a (selexipag, NS-304, ACT-293987), which is orally active and provides sustained plasma exposure of 12b. Compound 26a was developed for the treatment of pulmonary arterial hypertension and shown to reduce the risk of the composite morbidity/mortality end point in a phase 3 event-driven clinical trial. PMID:26291199

The behavioural effects in rats of the dopamine D2 receptoragonists, lisuride, B-HT 920 and SND 919, were variously influenced by pre-treatment with the selective alpha 2-adrenoceptor antagonist, idazoxan (2 mg/kg), depending on the nature of the effect in question and the doses of agonist employed. The influence of idazoxan on drug-induced stretching-yawning, penile erection, sedation, stereotyped behaviour, aggressiveness and mounting is described and tentatively interpreted in neurochemical terms, account being taken of the activity of respective alpha 2-adrenoceptor antagonist and dopamine receptoragonists used, at alpha 2-adrenoceptors and at different dopamine D2 receptor subtypes, pre- and postsynaptically located. PMID:7907024

Group II metabotropic glutamate receptor (mGluR) agonists have emerged as potential treatment drugs for schizophrenia and other neurological disorders, whereas the mechanisms involved remain elusive. Here we examined the effects of LY379268 (LY37) on the expression and trafficking of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunits GluA1 and GluA2 in prefrontal neurons. We show that LY37 significantly increased the surface and total expression of both GluA1 and GluA2 subunits in cultured prefrontal neurons and in vivo. This effect was mimicked by the selective mGluR2 agonist LY395756 and was blocked by mGluR2/3 antagonist LY341495. Moreover, we found that both GluA1 and GluA2 subunits were colocalized with PSD95 but not synapsin I, suggesting a postsynaptic localization. Consistently, treatment with LY37 significantly increased the amplitude, but not frequency, of miniature excitatory postsynaptic currents. Further, actinomycin-D blocked LY37's effects, suggesting a transcriptional regulation. In addition, application of glycogen synthase kinase-3beta (GSK-3β) inhibitor completely blocked LY37's effect on GluA2 surface expression, whereas GSK-3β inhibitor itself induced decreases in the surface and total protein levels of GluA1, but not GluA2 subunits. This suggests that GSK-3β differentially mediates GluA1 and GluA2 trafficking. Further, LY37 significantly increased the phosphorylation, but not total protein, of extracellular signal-regulated kinase 1/2 (ERK1/2). Neither ERK1/2 inhibitor PD98059 alone nor PD98059 combined with LY37 treatment induced changes in GluA1 or GluA2 surface expression or total protein levels. Our data thus suggest that mGluR2/3 agonist regulates postsynaptic AMPA receptors by affecting the synaptic trafficking of both GluA1 and GluA2 subunits and that the regulation is likely through ERK1/2 signaling in GluA1 and/or both ERK1/2 and GSK-3β signaling pathways in the GluA2 subunit. PMID:23593498

The plant stress hormone abscisic acid (ABA) is critical for several abiotic stress responses. ABA signaling is normally repressed by group-A protein phosphatases 2C (PP2Cs), but stress-induced ABA binds Arabidopsis PYR/PYL/RCAR (PYL) receptors, which then bind and inhibit PP2Cs. X-ray structures of several receptor-ABA complexes revealed a tunnel above ABA's 3' ring CH that opens at the PP2C binding interface. Here, ABA analogs with sufficiently long 3' alkyl chains were predicted to traverse this tunnel and block PYL-PP2C interactions. To test this, a series of 3'-alkylsulfanyl ABAs were synthesized with different alkyl chain lengths. Physiological, biochemical and structural analyses revealed that a six-carbon alkyl substitution produced a potent ABA antagonist that was sufficiently active to block multiple stress-induced ABA responses in vivo. This study provides a new approach for the design of ABA analogs, and the results validated structure-based design for this target class. PMID:24792952

The aim of this study was to reexamine the concept that gamma-hydroxybutyric acid (GHB) is a weak but selective agonist at gamma-aminobutyric acidB (GABAB) receptors, using binding experiments with several radioligands. Ki values of GHB were similar (approximately equal to 100 microM) in three agonist radioligand assays for GABAB receptors, [3H]baclofen (beta-para-chlorophenyl-gamma-aminobutyric acid), [3H]CGP 27492 (3-aminopropyl-phosphinic acid) and [3H]GABA, in the presence of the GABAA receptoragonist isoguvacine with rat cortical, cerebellar and hippocampal membranes. In competition experiments between GHB and the GABAB receptor antagonist, [3H]CGP 54626 (3-N [1-{(S)-3,4-dichlorophenyl}-ethylamino]-2-(S)-hydroxypropyl cyclo-hexylmethyl phosphinic acid), the IC50 values were significantly increased with 300 microM of 5'-guanyl-imidodiphosphate (Gpp(NH)p), which suggested that guanine nucleotide binding proteins (G-proteins) modulate GHB binding on GABAB receptors. The inhibition by GHB of [3H]CGP 27492 binding in cortical membranes was not altered in the presence of 0.3 or 3 mM of the two GHB dehydrogenase inhibitors, valproate and ethosuximide. Thus, GHB is not reconverted into GABA by GHB dehydrogenase. Taken together, the results of this study demonstrated that GHB is an endogenous weak but selective agonist at GABAB receptors. PMID:9083788

Cocaine dependence remains a challenging public health problem with relapse cited as a major determinant in its chronicity and severity. Environmental contexts and stimuli become reliably associated with its use leading to durable conditioned responses (‘cue reactivity') that can predict relapse as well as treatment success. Individual variation in the magnitude and influence of cue reactivity over behavior in humans and animals suggest that cue-reactive individuals may be at greater risk for the progression to addiction and/or relapse. In the present translational study, we investigated the contribution of variation in the serotonin (5-HT) 5-HT2Creceptor (5-HT2CR) system in individual differences in cocaine cue reactivity in humans and rodents. We found that cocaine-dependent subjects carrying a single nucleotide polymorphism (SNP) in the HTR2C gene that encodes for the conversion of cysteine to serine at codon 23 (Ser23 variant) exhibited significantly higher attentional bias to cocaine cues in the cocaine-word Stroop task than those carrying the Cys23 variant. In a model of individual differences in cocaine cue reactivity in rats, we identified that high cocaine cue reactivity measured as appetitive approach behavior (lever presses reinforced by the discrete cue complex) correlated with lower 5-HT2CR protein expression in the medial prefrontal cortex and blunted sensitivity to the suppressive effects of the selective 5-HT2CR agonist WAY163909. Our translational findings suggest that the functional status of the 5-HT2CR system is a mechanistic factor in the generation of vulnerability to cocaine-associated cues, an observation that opens new avenues for future development of biomarker and therapeutic approaches to suppress relapse in cocaine dependence. PMID:24618688

Muscarinic receptors in calf forebrain membranes can be identified by the specific binding of the radiolabelled antagonist [3H]dexetimide. These receptors (2.8 pM/mg protein) comprise two non-interconvertible subpopulations with respectively high and low agonist affinity but with the same antagonist affinity. For all the agonists tested the low affinity sites represent 85 +/- 5% of the total receptor population. 0.5% Digitonin solubilized extracts contain 0.8 pM muscarinic receptor/mg protein. In contrast with the membranes, these extracts contain only sites with low agonist affinity. The alkylating reagent N-ethylmaleimide causes an increase of the acetylcholine affinity for the low affinity sites in membranes as well as for the solubilized sites. This effect is time dependent until a maximal 3-fold increase in affinity is attained. The rate of N-ethylmaleimide action is enhanced by the concomitant presence of agonists. In contrast, N-ethylmaleimide does not affect antagonist binding. This suggests that agonists mediate a conformational change of both the membrane bound low affinity muscarinic sites and of the solubilized sites, resulting in their increased susceptibility towards NEM alkylation. PMID:6487351

Adiponectin, the adipose-derived hormone, plays an important role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. It has been shown that up-regulation of adiponectin or adiponectin receptor has a number of therapeutic benefits. Given that it is hard to convert the full size adiponectin protein into a viable drug, adiponectin receptoragonists could be designed or identified using high-throughput screening. Here, we report on the development of a two-step screening process to identify adiponectin agonists. First step, we developed a high throughput screening assay based on fluorescence polarization to identify adiponectin ligands. The fluorescence polarization assay reported here could be adapted to screening against larger small molecular compound libraries. A natural product library containing 10,000 compounds was screened and 9 hits were selected for validation. These compounds have been taken for the second-step in vitro tests to confirm their agonistic activity. The most active adiponectin receptor 1 agonists are matairesinol, arctiin, (-)-arctigenin and gramine. The most active adiponectin receptor 2 agonists are parthenolide, taxifoliol, deoxyschizandrin, and syringin. These compounds may be useful drug candidates for hypoadiponectin related diseases. PMID:23691032

Adiponectin, the adipose-derived hormone, plays an important role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. It has been shown that up-regulation of adiponectin or adiponectin receptor has a number of therapeutic benefits. Given that it is hard to convert the full size adiponectin protein into a viable drug, adiponectin receptoragonists could be designed or identified using high-throughput screening. Here, we report on the development of a two-step screening process to identify adiponectin agonists. First step, we developed a high throughput screening assay based on fluorescence polarization to identify adiponectin ligands. The fluorescence polarization assay reported here could be adapted to screening against larger small molecular compound libraries. A natural product library containing 10,000 compounds was screened and 9 hits were selected for validation. These compounds have been taken for the second-step in vitro tests to confirm their agonistic activity. The most active adiponectin receptor 1 agonists are matairesinol, arctiin, (-)-arctigenin and gramine. The most active adiponectin receptor 2 agonists are parthenolide, taxifoliol, deoxyschizandrin, and syringin. These compounds may be useful drug candidates for hypoadiponectin related diseases. PMID:23691032

The melanocortin receptors 3 and 4 control energy homeostasis, food-intake behavior, and correlated pathophysiological conditions. The melanocortin-4 receptor (MC4R) has been broadly investigated. In contrast, the knowledge related to physiological roles of the melanocortin-3 receptor (MC3R) is lacking because of the limited number of known MC3R selective ligands. Here, we report the design, synthesis, biological activity, conformational analysis, and docking with receptors of two potent and selective agonists at the human MC3 receptor. PMID:26599352

Kaempferol is a dietary flavonoid that is thought to function as a selective estrogen receptor modulator. In this study, we established that kaempferol also functions as an inverse agonist for estrogen-related receptors alpha and gamma (ERRalpha and ERRgamma). We demonstrated that kaempferol binds to ERRalpha and ERRgamma and blocks their interaction with coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha). Kaempferol also suppressed the expressions of ERR-target genes pyruvate dehydrogenase kinase 2 and 4 (PDK2 and PDK4). This evidence suggests that kaempferol may exert some of its biological effect through both estrogen receptors and estrogen-related receptors. PMID:19171140

Arachidonic acid (AA) is metabolized to epoxyeicosatrienoic acids (EETs) via cytochrome enzymes such as CYP 2C9, 2C8 and 2J2. EETs play a role in cardioprotection and regulation of blood pressure. Recently, adverse reactions such as sudden heart attack and fatal myocardial infarction were reported among patients taking angiotensin II receptor blockers (ARBs). As some ARBs have affinity for these CYP enzymes, metabolic inhibition of AA by ARBs is a possible cause for the increase in cardiovascular events. In this study, we quantitatively investigated the inhibitory effects of ARBs on the formation of EETs and further metabolites, dihydroxyeicosatrienoic acids (DHETs), from AA via CYP2C8. In incubations with recombinant CYP2C8 in vitro, the inhibitory effects were compared by measuring EETs and DHETs by HPLC-MS/MS. Inhibition of AA metabolism by ARBs was detected in a concentration-dependent manner with IC50 values of losartan (42.7 µM), telmisartan (49.5 µM), irbesartan (55.6 µM), olmesartan (66.2 µM), candesartan (108 µM), and valsartan (279 µM). Losartan, telmisartan and irbesartan, which reportedly accumulate in the liver and kidneys, have stronger inhibitory effects than other ARBs. The lower concentration of EETs leads to less protective action on the cardiovascular system and a higher incidence of adverse effects such as sudden heart attack and myocardial infarction in patients taking ARBs. PMID:26632190

Background Strychnine-sensitive glycine receptors in many adult forebrain regions consist of alpha2 + beta heteromeric channels. This subunit composition is distinct from the alpha1 + beta channels found throughout the adult spinal cord. Unfortunately, the pharmacology of forebrain alpha2beta receptors are poorly defined compared to 'neonatal' alpha2 homomeric channels or 'spinal' alpha1beta heteromers. In addition, the pharmacologic properties of native alpha2beta glycine receptors have been generally distinct from receptors produced by heterologous expression. To identify subtype-specific pharmacologic tools for the forebrain alpha2beta receptors, it is important to identify a heterologous expression system that closely resembles these native glycine-gated chloride channels. Results While exploring pharmacological properties of alpha2beta glycine receptors compared to alpha2-homomers, we found that distinct heterologous expression systems appeared to differentially influence partial agonist pharmacology. The β-amino acid taurine possessed 30–50% efficacy for alpha2-containing receptor isoforms when expressed in HEK 293 cells. However, taurine efficacy was dramatically reduced in L-cell fibroblasts. Similar results were obtained for β-alanine. The efficacy of these partial agonists was also strongly reduced by the beta subunit. There were no significant differences in apparent strychnine affinity values calculated from concentration-response data between expression systems or subunit combinations. Nor did relative levels of expression correlate with partial agonist efficacy when compared within or between several different expression systems. Finally, disruption of the tubulin cytoskeleton reduced the efficacy of partial agonists in a subunit-dependent, but system-independent, fashion. Conclusions Our results suggest that different heterologous expression systems can dramatically influence the agonist pharmacology of strychnine-sensitive glycine receptors. In

Background Resveratrol, a naturally occurring stilbene, has been categorized as a phytoestrogen due to its ability to compete with natural estrogens for binding to estrogen receptor alpha (ERα) and modulate the biological responses exerted by the receptor. Biological effects of resveratrol (RES) on estrogen receptor alpha (ERα) remain highly controversial, since both estrogenic and anti-estrogenic properties were observed. Results Here, we provide insight into the structural basis of the agonist/antagonist effects of RES on ERα ligand binding domain (LBD). Using atomistic simulation, we found that RES bound ERα monomer in antagonist conformation, where Helix 12 moves away from the ligand pocket and orients into the co-activator binding groove of LBD, is more stable than RES bound ERα in agonist conformation, where Helix 12 lays over the ligand binding pocket. Upon dimerization, the agonistic conformation of RES-ERα dimer becomes more stable compared to the corresponding monomer but still remains less stable compared to the corresponding dimer in antagonist conformation. Interestingly, while the binding pocket and the binding contacts of RES to ERα are similar to those of pure agonist diethylstilbestrol (DES), the binding energy is much less and the hydrogen bonding contacts also differ providing clues for the partial agonistic character of RES on ERα. Conclusions Our Molecular Dynamics simulation of RES-ERα structures with agonist and antagonist orientations of Helix 12 suggests RES action is more similar to Selective Estrogen Receptor Modulator (SERM) opening up the importance of cellular environment and active roles of co-regulator proteins in a given system. Our study reveals that potential co-activators must compete with the Helix 12 and displace it away from the activator binding groove to enhance the agonistic activity. PMID:24160181

Agonist concentration-response relationships at nicotinic postsynaptic receptors were established by measuring /sup 86/Rb/sup +/ efflux from acetylcholine receptor rich native Torpedo membrane vesicles under three different conditions: (1) integrated net ion efflux (in 10 s) from untreated vesicles, (2) integrated net efflux from vesicles in which most acetylcholine sites were irreversibly blocked with ..cap alpha..-bungarotoxin, and (3) initial rates of efflux (5-100 ms) from vesicles that were partially blocked with ..cap alpha..-bungarotoxin. Exposure to acetylcholine, carbamylcholine, suberyldicholine, phenyltrimethylammonium, or (-)-nicotine over 10/sup 8/-fold concentration ranges results in bell-shaped ion flux response curves due to stimulation of acetylcholine receptor channel opening at low concentrations and inhibition of channel function at 60-2000 times higher concentrations. Concentrations of agonists that inhibit their own maximum /sup 86/Rb/sup +/ efflux by 50% (K/sub B/ values) are 110, 211, 3.0, 39, and 8.9 mM, respectively, for the agonists listed above. For acetylcholine and carbamylcholine, K/sub B/ values determined from both 10-s and 15-ms efflux measurements are the same, indicating that the rate of agonist-induced desensitization increases to maximum at concentrations lower than those causing self-inhibition. For all partial and full agonists studied, Hill coefficients for self-inhibition are close to 1.0. Concentrations of agonists up to 8 times K/sub B/ did not change the order parameter reported by a spin-labeled fatty acid incorporated in Torpedo membranes. The authors conclude that agonist self-inhibition cannot be attributed to a general nonspecific membrane perturbation. Instead, these results are consistent with a saturable site of action either at the lipid-protein interface or on the acetylcholine receptor protein itself.

Retinoic acid signaling regulates several biological events, including myogenesis. We previously found that retinoic acid receptor γ (RARγ) agonist blocks heterotopic ossification, a pathological bone formation that mostly occurs in the skeletal muscle. Interestingly, RARγ agonist also weakened deterioration of muscle architecture adjacent to the heterotopic ossification lesion, suggesting that RARγ agonist may oppose skeletal muscle damage. To test this hypothesis, we generated a critical defect in the tibialis anterior muscle of 7-week-old mice with a cautery, treated them with RARγ agonist or vehicle corn oil, and examined the effects of RARγ agonist on muscle repair. The muscle defects were partially repaired with newly regenerating muscle cells, but also filled with adipose and fibrous scar tissue in both RARγ-treated and control groups. The fibrous or adipose area was smaller in RARγ agonist-treated mice than in the control. In addition, muscle repair was remarkably delayed in RARγ-null mice in both critical defect and cardiotoxin injury models. Furthermore, we found a rapid increase in retinoid signaling in lacerated muscle, as monitored by retinoid signaling reporter mice. Together, our results indicate that endogenous RARγ signaling is involved in muscle repair and that selective RARγ agonists may be beneficial to promote repair in various types of muscle injuries. PMID:26205250

The NMDA receptor (R) plays important roles in brain physiology and pathology as an ion channel. Here we examine the ion flow-independent coupling of agonist to the NMDAR cytoplasmic domain (cd). We measure FRET between fluorescently tagged cytoplasmic domains of GluN1 subunits of NMDARs expressed in neurons. Different neuronal compartments display varying levels of FRET, consistent with different NMDARcd conformations. Agonist binding drives a rapid and transient ion flow-independent reduction in FRET between GluN1 subunits within individual NMDARs. Intracellular infusion of an antibody targeting the GluN1 cytoplasmic domain blocks agonist-driven FRET changes in the absence of ion flow, supporting agonist-driven movement of the NMDARcd. These studies indicate that extracellular ligand binding to the NMDAR can transmit conformational information into the cell in the absence of ion flow. PMID:26553997

In the search for potent and selective human beta3-adrenergic receptor (AR) agonists as potential drugs for the treatment of obesity and noninsulin-dependent (type II) diabetes, a novel series of acetanilide-based analogues were prepared and their biological activities were evaluated at the human beta3-, beta2-, and beta1-ARs. Among these compounds, 2-pyridylacetanilide (2f), pyrimidin-2-ylacetanilide (2u), and pyrazin-2-ylacetanilide (2v) derivatives exhibited potent agonistic activity at the beta3-AR with functional selectivity over the beta1- and beta2-ARs. In particular, compound 2u was found to be the most potent and selective beta3-AR agonist with an EC(50) value of 0.11 microM and no agonistic activity for either the beta1- or beta2-AR. In addition, 2f, 2u, and 2v showed significant hypoglycemic activity in a rodent diabetic model. PMID:19232786

Toll-like receptor (TLR) ligation activates both the innate and adaptive immune systems, and plays an important role in antiviral and anti-tumor immunity. Therefore, a significant amount of effort has been devoted to exploit the therapeutic potential of TLR agonists. Depending on the therapeutic purpose, either as adjuvants to vaccine, chemotherapy or standalone therapy, TLR agonists have been administered via different routes. Both preclinical and clinical studies have suggested that the route of administration has significant effects on pharmacokinetics, and that understanding these effects is critical to the success of TLR agonist drug development. This article will summarize the pharmacokinetics of TLR agonists with different administration routes, with an emphasis on clinical studies of TLR ligands in oncologic applications. PMID:21643519

Toll-like receptor (TLR) ligation activates both the innate and adaptive immune systems, and plays an important role in antiviral and anti-tumor immunity. Therefore, a significant amount of effort has been devoted to exploit the therapeutic potential of TLR agonists. Depending on the therapeutic purpose, either as adjuvants to vaccine, chemotherapy or standalone therapy, TLR agonists have been administered via different routes. Both preclinical and clinical studies have suggested that the route of administration has significant effects on pharmacokinetics, and that understanding these effects is critical to the success of TLR agonist drug development. This article will summarize the pharmacokinetics of TLR agonists with different administration routes, with an emphasis on clinical studies of TLR ligands in oncologic applications. PMID:21643519

The NMDA receptor (R) plays important roles in brain physiology and pathology as an ion channel. Here we examine the ion flow-independent coupling of agonist to the NMDAR cytoplasmic domain (cd). We measure FRET between fluorescently tagged cytoplasmic domains of GluN1 subunits of NMDARs expressed in neurons. Different neuronal compartments display varying levels of FRET, consistent with different NMDARcd conformations. Agonist binding drives a rapid and transient ion flow-independent reduction in FRET between GluN1 subunits within individual NMDARs. Intracellular infusion of an antibody targeting the GluN1 cytoplasmic domain blocks agonist-driven FRET changes in the absence of ion flow, supporting agonist-driven movement of the NMDARcd. These studies indicate that extracellular ligand binding to the NMDAR can transmit conformational information into the cell in the absence of ion flow. PMID:26553997

At present, molecular cloning and pharmacological studies have delineated three human alpha 2-adrenergic receptor (alpha 2AR) subtypes, alpha 2C10, alpha 2C4, and alpha 2C2. Assignment of the alpha 2AR subtypes to specific functions has been limited by an unclear definition of tissue alpha 2AR expression outside of the central nervous system. It has been suggested that alpha 2C4 expression is confined to the brain, that alpha 2C2 expression is only in the liver and kidney, and that there is nearly ubiquitous expression of alpha 2C10. However, this is based on studies of a limited number of rat tissues or on studies using non-species-specific approaches. Therefore, to define alpha 2C10, alpha 2C4, and alpha 2C2 tissue expression, we used reverse transcription of total RNA isolated from 20 human tissues, followed by amplification of alpha 2AR cDNA using the polymerase chain reaction. This technique provided two advantages: high sensitivity and, with the use of subtype-specific oligonucleotide primers and probes, differentiation between the alpha 2AR subtypes. The tissues studied were aorta, vena cava, heart (epicardium and endocardium), lung, skeletal muscle, liver, pancreas (head and tail), fat (perinephric and subcutaneous), kidney (cortex and medulla), prostate, stomach, ileum, jejunum, colon, adrenal gland, and spleen. We found that the majority of these tissues expressed alpha 2C10, with the exceptions being the head of the pancreas, subcutaneous fat, colon, and spleen. In marked distinction to other studies, however, we found a prolific expression of the alpha 2C4 and alpha 2C2 subtypes. Expression of alpha 2C4 was found in all tissues with the exception of liver, fat, stomach, and colon, and a virtually ubiquitous expression of alpha 2C2 was found, with the exception of epicardium. Of all tissues studied, only colon and subcutaneous fat expressed a single alpha 2AR subtype, which was alpha 2C2. Thus, the alpha 2AR subtypes do not have a confined expression but

Serotonin-1A receptors may play a role in the pathophysiology of depression and suicide. In postmortem brain tissue, agonist binding to serotonin-1A receptors is reportedly increased or unchanged in depression or suicide, while neuroimaging studies report a decrease in antagonist binding to these receptors in subjects with depression. In this study, both agonist and antagonist radioligand binding to serotonin-1A receptors were examined in postmortem orbitofrontal cortex from subjects with major depressive disorder (MDD). Brain tissue was collected at autopsy from 11 subjects with MDD and 11 age- and gender-matched normal control subjects. Two depressed subjects had a recent psychoactive substance use disorder. Six subjects with MDD had a prescription for an antidepressant drug in the last month of life, and, of these six, postmortem bloods from only two subjects tested positive for an antidepressant drug. There was no significant difference between cohorts for age, postmortem interval or tissue pH. The receptoragonist [3H]8-OH-DPAT or the antagonist [3H]MPPF were used to autoradiographically label serotonin-1A receptors in frozen sections from cytoarchitectonically-defined left rostral orbitofrontal cortex (area 47). There was no significant difference between depressed and control subjects in agonist binding to serotonin-1A receptors. However, antagonist binding was significantly decreased in outer layers of orbitofrontal cortex in MDD. This observation in postmortem tissue confirms reports using an antagonist radioligand in living subjects with depression. Decreased antagonist binding to serotonin-1A receptors in outer layers of orbitofrontal cortex suggests diminished receptor signaling and may be linked to corresponding neuronal changes detected previously in these depressed subjects. PMID:19215942

Background Methadone is a unique µ-opioid receptoragonist. Although several researchers have insisted that the pharmacological effects of methadone are mediated through the blockade of NMDA receptor, the underlying mechanism by which methadone exerts its distinct pharmacological effects compared to those of other µ-opioid receptoragonists is still controversial. In the present study, we further investigated the pharmacological profile of methadone compared to those of fentanyl and morphine as measured mainly by the discriminative stimulus effect and in vitro assays for NMDA receptor binding, µ-opioid receptor-internalization, and µ-opioid receptor-mediated β-arrestin recruitment. Results We found that fentanyl substituted for the discriminative stimulus effects of methadone, whereas a relatively high dose of morphine was required to substitute for the discriminative stimulus effects of methadone in rats. Under these conditions, the non-competitive NMDA receptor antagonist MK-801 did not substitute for the discriminative stimulus effects of methadone. In association with its discriminative stimulus effect, methadone failed to displace the receptor binding of MK801 using mouse brain membrane. Methadone and fentanyl, but not morphine, induced potent µ-opioid receptor internalization accompanied by the strong recruitment of β-arrestin-2 in µ-opioid receptor-overexpressing cells. Conclusions These results suggest that methadone may, at least partly, produce its pharmacological effect as a β-arrestin-biased µ-opioid receptoragonist, similar to fentanyl, and NMDA receptor blockade is not the main contributor to the pharmacological profile of methadone. PMID:27317580

The Gi-coupled A3 adenosine receptor (A3AR) mediates anti-inflammatory, anticancer and anti-ischemic protective effects. The receptor is overexpressed in inflammatory and cancer cells, while low expression is found in normal cells, rendering the A3AR as a potential therapeutic target. Highly selective A3AR agonists have been synthesized and molecular recognition in the binding site has been characterized. The present review summarizes preclinical and clinical human studies demonstrating that A3AR agonists induce specific anti-inflammatory and anticancer effects via a molecular mechanism that entails modulation of the Wnt and the NF-κB signal transduction pathways. Currently, A3AR agonists are being developed for the treatment of inflammatory diseases including rheumatoid arthritis and psoriasis; ophthalmic diseases such as dry eye syndrome and glaucoma; liver diseases such as hepatocellular carcinoma and hepatitis. PMID:22033198

Background and Purpose Artificial agonists may have higher efficacy for receptor activation than the physiological agonist. Until now, such ‘superagonism’ has rarely been reported for GPCRs. Iperoxo is an extremely potent muscarinic receptoragonist. We hypothesized that iperoxo is a ‘superagonist’. Experimental Approach Signalling of iperoxo and newly synthesized structural analogues was compared with that of ACh at label-free M2 muscarinic receptors applying whole cell dynamic mass redistribution, measurement of G-protein activation, evaluation of cell surface agonist binding and computation of operational efficacies. Key Results In CHO-hM2 cells, iperoxo significantly exceeds ACh in Gi/Gs signalling competence. In the orthosteric loss-of-function mutant M2-Y1043.33A, the maximum effect of iperoxo is hardly compromised in contrast to ACh. ‘Superagonism’ is preserved in the physiological cellular context of MRC-5 human lung fibroblasts. Structure–signalling relationships including iperoxo derivatives with either modified positively charged head group or altered tail suggest that ‘superagonism’ of iperoxo is mechanistically based on parallel activation of the receptor protein via two orthosteric interaction points. Conclusion and Implications Supraphysiological agonist efficacy at muscarinic M2 ACh receptors is demonstrated for the first time. In addition, a possible underlying molecular mechanism of GPCR ‘superagonism’ is provided. We suggest that iperoxo-like orthosteric GPCR activation is a new avenue towards a novel class of receptor activators. Linked Article This article is commented on by Langmead and Christopoulos, pp. 353–356 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.12142 PMID:23062057

In 1991, a potent 61 amino acid vasodilator peptide, named maxadilan, was isolated from the salivary glands of the sand fly. Subsequently, it was shown that this peptide specifically and potently activated the mammalian PAC1 receptor, one of the three receptors for PACAP. These studies and the link between maxadilan and leishmaniasis are discussed. PMID:17681401

Neural function within the medial prefrontal cortex (mPFC) regulates normal cognition, attention and impulse control, implicating neuroregulatory abnormalities within this region in mental dysfunction related to schizophrenia, depression and drug abuse. Both serotonin -2A (5-HT2A) and -2C (5-HT2C) receptors are known to be important in neuropsychiatric drug action and are distributed throughout the mPFC. However, their interactive role in serotonergic cortical regulation is poorly understood. While the main signal transduction mechanism for both receptors is stimulation of phosphoinositide production, they can have opposite effects downstream. 5-HT2A versus 5-HT2Creceptor activation oppositely regulates behavior and can oppositely affect neurochemical release within the mPFC. These distinct receptor effects could be caused by their differential cellular distribution within the cortex and/or other areas. It is known that both receptors are located on GABAergic and pyramidal cells within the mPFC, but it is not clear whether they are expressed on the same or different cells. The present work employed immunofluorescence with confocal microscopy to examine this in layers V-VI of the prelimbic mPFC. The majority of GABA cells in the deep prelimbic mPFC expressed 5-HT2Creceptor immunoreactivity. Furthermore, most cells expressing 5-HT2Creceptor immunoreactivity notably co-expressed 5-HT2A receptors. However, 27% of 5-HT2Creceptor immunoreactive cells were not GABAergic, indicating that a population of prelimbic pyramidal projection cells could express the 5-HT2Creceptor. Indeed, some cells with 5-HT2C and 5-HT2A receptor co-labeling had a pyramidal shape and were expressed in the typical layered fashion of pyramidal cells. This indirectly demonstrates that 5-HT2C and 5-HT2A receptors may be commonly co-expressed on GABAergic cells within the deep layers of the prelimbic mPFC and perhaps co-localized on a small population of local pyramidal projection cells. Thus a

Opioids are the most effective and widely used drugs for pain treatment. Morphine is an archetypal opioid and is an opioid receptoragonist. Unfortunately, the clinical usefulness of morphine is limited by adverse effects such as analgesic tolerance and addiction. Therefore, it is important to study the development of novel opioid agonists as part of pain control. The analgesic effects of opioids are mediated by three opioid receptors, namely opioid μ-, δ-, and κ-receptors. They belong to the G protein-coupled receptor superfamily and are coupled to Gi proteins. In the present study, we developed a ligand screening system to identify novel opioid μ-receptoragonists that measures [(35)S]GTPγS binding to cell membrane fractions prepared from the fat body of transgenic silkworms expressing μ-receptor-Gi1α fusion protein. We screened the RIKEN Natural Products Depository (NPDepo) chemical library, which contains 5848 compounds, and analogs of hit compounds. We successfully identified a novel, structurally unique compound, that we named GUM1, with agonist activity for the opioid μ-receptor (EC50 of 1.2 µM). The Plantar Test (Hargreaves' Method) demonstrated that subcutaneous injection of 3mg/kg of GUM1 into wild-type rats significantly extended latency time. This extension was also observed in a rat model of morphine tolerance and was inhibited by pre-treatment of naloxone. The unique molecular skeleton of GUM1 makes it an attractive molecule for further ligand-opioid receptor binding studies. PMID:26476280

Long-acting glucagon-like peptide-1 receptor (GLP-1R) agonists have both glucose- and weight-lowering effects. The brain is poised to mediate both of these actions since GLP-1Rs are present in key areas known to control weight and glucose. Although some research has been performed on the effects of ...

We examined whether the cannabinoid receptoragonist WIN55,212-2 (WIN; 5 [mu]g/side) microinjected into the hippocampus or the amygdala would differentially affect memory processes in a neutral vs. an aversive task. In the aversive contextual fear task, WIN into the basolateral amygdala impaired fear acquisition/consolidation, but not retrieval.…

The melanocortin receptor 4 (MC4R) plays a major role in body weight regulation and its agonist MTII has been widely used to study the role of MC4Rs in energy expenditure promotion and feeding reduction. Unexpectedly, we observed that intraperitoneal (i.p.) administration of MTII induced a rapid red...

The chemokine CXCL12 (SDF-1) and its cognate receptor CXCR4 are involved in a large number of physiological processes including HIV-1 infectivity, inflammation, tumorigenesis, stem cell migration, and autoimmune diseases. While previous efforts have identified a number of CXCR4 antagonists, there have been no small molecule agonists reported. Herein, we describe the identification of a novel series of CXCR4 modulators, including the first small molecules to display agonist behavior against this receptor, using a combination of structure- and ligand-based virtual screening. These agonists produce robust calcium mobilization in human melanoma cell lines which can be blocked by the CXCR4-selective antagonist AMD3100. We also demonstrate the ability of these new agonists to induce receptor internalization, ERK activation, and chemotaxis, all hallmarks of CXCR4 activation. Our results describe a new series of biologically relevant small molecules that will enable further study of the CXCR4 receptor and may contribute to the development of new therapeutics. PMID:27456816

A graphics model of the human 5-HT6 receptor was constructed and automated docking studies were performed. The model suggests that 5-HT6 antagonist arylsulfonyltryptamines might bind differently than that of the agonist serotonin. Furthermore, the model explains many of the empirical results from our previous structure-affinity studies. PMID:15357994

The chemokine CXCL12 (SDF-1) and its cognate receptor CXCR4 are involved in a large number of physiological processes including HIV-1 infectivity, inflammation, tumorigenesis, stem cell migration, and autoimmune diseases. While previous efforts have identified a number of CXCR4 antagonists, there have been no small molecule agonists reported. Herein, we describe the identification of a novel series of CXCR4 modulators, including the first small molecules to display agonist behavior against this receptor, using a combination of structure- and ligand-based virtual screening. These agonists produce robust calcium mobilization in human melanoma cell lines which can be blocked by the CXCR4-selective antagonist AMD3100. We also demonstrate the ability of these new agonists to induce receptor internalization, ERK activation, and chemotaxis, all hallmarks of CXCR4 activation. Our results describe a new series of biologically relevant small molecules that will enable further study of the CXCR4 receptor and may contribute to the development of new therapeutics. PMID:27456816

Background and Purpose Since the identification of the histamine H4 receptor, several ligands activating this receptor have been described and more compounds are in development. These ligands are well characterized in pharmacological assays, including radioligand competition binding studies, GTPγS and GTPase assays. In most cases, these experiments are performed in transfected cell lines, expressing unnaturally high levels of target receptors and G-protein signalling components. In this study we investigated the specific properties of H4 receptor ligands in native cells. Experimental Approach Histamine and five different H4 receptoragonists – 4-methylhistamine, UR-PI376, clobenpropit, VUF8430 and ST-1006 – were characterized in freshly isolated human monocytes. The ligands (10 nM–10 μM) were tested as inhibitors of IL-12p70 secretion from human monocytes and the effects of the H2 receptor antagonist ranitidine and the H4 receptor antagonist JNJ7777120 on their action was investigated. Key Results Histamine and all the tested agonists reduced IL-12p70 secretion into monocyte supernatants by 40–70%. The potencies varied with pEC50 values ranging from 5.7 to 6.9, depending on the agonist used. All potencies were lower than those determined in the original investigations of the compounds. Pretreatment of monocytes with H2 or H4 receptor antagonists showed that some H4 receptor ligands also had low activity at the H2 receptor. Conclusions and Implications Our study demonstrates discrepancies between the potencies obtained from assays in transfected cell lines and assays in native human cells, indicating the importance of evaluating H4 receptor ligands in native cells. Linked Articles This article is part of a themed issue on Histamine Pharmacology Update. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2013.170.issue-1 PMID:23638754

The authors previous experiments in membranes prepared from rat heart and brain led them to suggest that the binding of agonist to the muscarinic receptors and to the Na/sup +/ channels is a coupled event mediated by guanine nucleotide binding protein(s) (G-protein(s)). These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of (/sup 3/H) acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of (/sup 3/H)batrachotoxin to Na/sup +/ channel(s) in the presence of the muscarinic agonists; and (iii) muscarinically induced /sup 22/Na/sup +/ uptake in the presence and absence of tetrodotoxin, which blocks Na/sup +/ channels. The findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na/sup +/channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components-receptor, G-protein, and Na/sup +/ channel-is such that at resting potential the muscarinic receptor induces opening of Na/sup +/ channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues.

The ability of benzodiazepine receptor partial agonists to exhibit full efficacy in preclinical anxiolytic tests, in conjunction with initial clinical results, has suggested the possibility of a reduced clinical side-effect profile compared to benzodiazepine receptor full agonists like diazepam. Because punished behavior of pigeons has been useful in detecting effects of novel anxiolytic drugs, effects of imidazobenzodiazepine and beta-carboline benzodiazepine receptor partial agonists and some related compounds were evaluated in this species. The abilities of these compounds to substitute for the discriminative stimulus effects of the full agonists midazolam also was determined. Intrinsic efficacy was assessed by the degree to which gamma-aminobutyric acid increased ligand potency to displace [(3)H]Ro15-1788 (flumazinil) from membranes of pigeon cerebrum, and ranged from full agonist-like efficacy (Ro 19-5470; 7-(3-cyclopropyl-1,2,4-oxodiazol-5-yl)-5,6-dihydro-5-methyl-4H- imidazo[1,5a]-thieno[3,2-f]diazin-4-one) to minimal gamma-aminobutyric acid potentiations close to that of the antagonist flumazenil (abecarnil and Ro 41-7812; 7-chloro-4,5-dihydro-3-(3-hydroxy-1-propynyl)-5-methyl-6H-imidazo[1,5-a] -[1,4 ]benzodiazepine-6-one). Punished responding was increased markedly by midazolam and by all partial agonists, except Ro 41-7812 and Ro 42-8773 (7-chloro-3-[3-(cyclopropylmethoxy)-1-propynyl]-4,5-dihyro-5 -methyl-6H-imidaz o[1,5-a][1,4]benzodiazepine-6-one), at doses that did not affect nonpunished responding. In contrast to the full substitution generally observed in mammals, all of the partial agonists produced incomplete substitution (40-70%) in the midazolam drug discrimination procedure in pigeons. A positive relationship was observed between the degree of substitution and intrinsic efficacy. The benzodiazepine antagonists, flumazenil and ZK 93,426 (ethyl-5-isopropoxy-4-methoxymethyl-beta-carboline-3-carboxylate), neither increased punished responding nor

To characterize the structural requirements for ligand orientation compatible with activation of the Torpedo nicotinic acetylcholine receptor (nAChR), we used Cys mutagenesis in conjunction with sulfhydryl-reactive reagents to tether primary or quaternary amines at defined positions within the agonist binding site of nAChRs containing mutant alpha- or gamma-subunits expressed in Xenopus oocytes. 4-(N-Maleimido)benzyltrimethylammonium and 2-aminoethylmethanethiosulfonate acted as irreversible antagonists when tethered at alphaY93C, alphaY198C, or gammaE57C, as well as at alphaN94C (2-aminoethylmethanethiosulfonate only). [2-(Trimethylammonium)-ethyl]-methanethiosulfonate (MTSET), which attaches thiocholine to binding site Cys, also acted as an irreversible antagonist when tethered at alphaY93C, alphaN94C, or gammaE57C. However, MTSET modification of alphaY198C resulted in prolonged activation of the nAChR not reversible by washing but inhibitable by subsequent exposure to non-competitive antagonists. Modification of alphaY198C (or any of the other positions tested) by [(trimethylammonium)methyl]methanethiosulfonate resulted only in irreversible inhibition, while modification of alphaY198C by [3-(trimethylammonium)propyl]methanethiosulfonate resulted in irreversible activation of nAChR, but at lower efficacy than by MTSET. Thus changing the length of the tethering arm by less than 1 A in either direction markedly effects the ability of the covalent trimethylammonium to activate the nAChR, and agonist activation depends on a very selective orientation of the quaternary ammonium within the agonist binding site. PMID:10777557

One of the hallmarks of alcohol dependence is the presence of a withdrawal syndrome during abstinence, which manifests as physical craving for alcohol accompanied by subjective feelings of anxiety. Using a model of chronic intermittent ethanol (CIE) vapor in mice, we investigated the role of serotonin2creceptor (5HT2c-R) signaling in the BNST as a neural substrate underlying ethanol-induced anxiety during withdrawal. Mice were subjected to a 5-day CIE regimen of 16 h of ethanol vapor exposure followed by an 8 h "withdrawal" period between exposures. After the 5th and final exposure, mice were withdrawn for 24 h or 1 week before experiments began. Anxiety-like behavior was assessed in the social approach, light dark, and open field tests with mice showing deficits in social, but not general anxiety-like behavior that was alleviated by pretreatment with the 5HT2c-R antagonist SB 242,084 (3 mg/kg, i.p.) 24 h and 1 week post-CIE. Using immunohistochemistry and whole cell patch clamp electrophysiology, we also found that CIE increased FOS-IR and enhanced neuronal excitability in the ventral BNST (vBNST) 24 h into withdrawal in a 5HT2c-R dependent manner. This enhanced excitability persisted for 1 week post-CIE. We also found that mCPP, a 5HT2c/b agonist, induced a more robust depolarization in cells of the vBNST in CIE mice, confirming that 5HT2c-R signaling is upregulated in the vBNST following CIE. Taken together, these results suggest that CIE upregulates 5HT2c-R signaling in the vBNST, leading to increased excitability. This enhanced excitability of the vBNST may drive increased anxiety-like behavior during ethanol withdrawal. PMID:25229718

Background: Desensitization and blockade of 5-HT2Creceptors (5-HT2CR) have long been thought to be central in the therapeutic action of antidepressant drugs. However, besides behavioral pharmacology studies, there is little in vivo data documenting antidepressant-induced 5-HT2CR desensitization in specific brain areas. Methods: Mice lacking the 5-HT reuptake carrier (5-HTT-/-) were used to model the consequences of chronic 5-HT reuptake inhibition with antidepressant drugs. The effect of this mutation on 5-HT2CR was evaluated at the behavioral (social interaction, novelty-suppressed feeding, and 5-HT2CR–induced hypolocomotion tests), the neurochemical, and the cellular (RT-qPCR, mRNA editing, and c-fos–induced expression) levels. Results: Although 5-HTT-/- mice had an anxiogenic profile in the novelty-suppressed feeding test, they displayed less 5-HT2CR–mediated anxiety in response to the agonist m-chlorophenylpiperazine in the social interaction test. In addition, 5-HT2CR–mediated inhibition of a stress-induced increase in 5-HT turnover, measured in various brain areas, was markedly reduced in 5-HTT-/- mutants. These indices of tolerance to 5-HT2CR stimulation were associated neither with altered levels of 5-HT2CR protein and mRNA nor with changes in pre-mRNA editing in the frontal cortex. However, basal c-fos mRNA production in cells expressing 5-HT2CR was higher in 5-HTT-/- mutants, suggesting an altered basal activity of these cells following sustained 5-HT reuptake carrier inactivation. Furthermore, the increased c-fos mRNA expression in 5-HT2CR–like immune-positive cortical cells observed in wild-type mice treated acutely with the 5-HT2CR agonist RO-60,0175 was absent in 5-HTT-/- mutants. Conclusions: Such blunted responsiveness of the 5-HT2CR system, observed at the cell signaling level, probably contributes to the moderation of the anxiety phenotype in 5-HTT-/- mice. PMID:25522398

Although central serotonergic systems are known to influence responses to noxious stimuli, mechanisms underlying serotonergic modulation of pain responses are unclear. We proposed that serotonin 2Creceptors (5-HT2CRs), which are expressed within brain regions implicated in sensory and affective responses to pain, contribute to the serotonergic modulation of pain responses. In mice constitutively lacking 5-HT2CRs (2CKO mice) we found normal baseline sensory responses to noxious thermal, mechanical and chemical stimuli. In contrast, 2CKO mice exhibited a selective enhancement of affect-related ultrasonic afterdischarge vocalizations in response to footshock. Enhanced affect-related responses to noxious stimuli were also exhibited by 2CKO mice in a fear-sensitized startle assay. The extent to which a brief series of unconditioned footshocks produced enhancement of acoustic startle responses was markedly increased in 2CKO mice. As mesolimbic dopamine pathways influence affective responses to noxious stimuli, and these pathways are disinhibited in 2CKO mice, we examined the sensitivity of footshock-induced enhancement of startle to dopamine receptor blockade. Systemic administration of the dopamine D2/D3 receptor antagonist raclopride selectively reduced footshock-induced enhancement of startle without influencing baseline acoustic startle responses. We propose that 5-HT2CRs regulate affective behavioral responses to unconditioned aversive stimuli through mechanisms involving the disinhibition of ascending dopaminergic pathways. PMID:26630489

Serotonin2C (5-HT(2C)) receptors act in the basal ganglia, a group of sub-cortical structures involved in motor behavior, where they are thought to modulate oral activity and participate in iatrogenic motor side-effects in Parkinson's disease and Schizophrenia. Whether abnormal movements initiated by 5-HT(2C) receptors are directly consequent to dysfunctions of the motor circuit is uncertain. In the present study, we combined behavioral, immunohistochemical and extracellular single-cell recordings approaches in rats to investigate the effect of the 5-HT(2C) agonist Ro-60-0175 respectively on orofacial dyskinesia, the expression of the marker of neuronal activity c-Fos in basal ganglia and the electrophysiological activity of substantia nigra pars reticulata (SNr) neuron connected to the orofacial motor cortex (OfMC) or the medial prefrontal cortex (mPFC). The results show that Ro-60-0175 (1 mg/kg) caused bouts of orofacial movements that were suppressed by the 5-HT(2C) antagonist SB-243213 (1 mg/kg). Ro-60-0175 (0.3, 1, 3 mg/kg) dose-dependently enhanced Fos expression in the striatum and the nucleus accumbens. At the highest dose, it enhanced Fos expression in the subthalamic nucleus, the SNr and the entopeduncular nucleus but not in the external globus pallidus. However, the effect of Ro-60-0175 was mainly associated with associative/limbic regions of basal ganglia whereas subregions of basal ganglia corresponding to sensorimotor territories were devoid of Fos labeling. Ro-60-0175 (1-3 mg/kg) did not affect the electrophysiological activity of SNr neurons connected to the OfMC nor their excitatory-inhibitory-excitatory responses to the OfMC electrical stimulation. Conversely, Ro-60-0175 (1 mg/kg) enhanced the late excitatory response of SNr neurons evoked by the mPFC electrical stimulation. These results suggest that oral dyskinesia induced by 5-HT(2C) agonists are not restricted to aberrant signalling in the orofacial motor circuit and demonstrate discrete

Chemotherapy is used to treat numerous cancers including melanoma. However, its effectiveness in clinical settings is often hampered by various mechanisms. Previous studies have demonstrated that prooxidative stressor-mediated generation of oxidized lipids with platelet-activating factor-receptor (PAF-R) agonistic activity induces systemic immunosuppression that augments the growth of experimental melanoma tumors. We have recently shown that treatment of murine B16F10 melanoma cells in vitro or tumors implanted into syngeneic mice and treated intratumorally with various chemotherapeutic agents generated PAF-R agonists in a process blocked by antioxidants. Notably, these intratumoral chemotherapy-generated PAF-R agonists augmented the growth of secondary (untreated) tumors in a PAF-R dependent manner. As both localized and systemic chemotherapies are used based on tumor localization/stage and metastases, the current studies were sought to determine effects of PAF-R agonists on systemic chemotherapy against experimental melanoma. Here, we show that systemic chemotherapy with etoposide (ETOP) attenuates the growth of melanoma tumors when given subsequent to the tumor cell implantation. Importantly, this ETOP-mediated suppression of melanoma tumor growth was blocked by exogenous administration of a PAF-R agonist, CPAF. These findings indicate that PAF-R agonists not only negatively affect the ability of localized chemotherapy but also compromise the efficacy of systemic chemotherapy against murine melanoma. PMID:25922565

Airway remodeling is a hallmark feature of asthma and chronic obstructive pulmonary disease. Clinical studies and animal models have demonstrated increased airway smooth muscle (ASM) mass, and ASM thickness is correlated with severity of the disease. Current medications control inflammation and reverse airway obstruction effectively but have limited effect on remodeling. Recently we identified the expression of bitter taste receptors (TAS2R) on ASM cells, and activation with known TAS2R agonists resulted in ASM relaxation and bronchodilation. These studies suggest that TAS2R can be used as new therapeutic targets in the treatment of obstructive lung diseases. To further establish their effectiveness, in this study we aimed to determine the effects of TAS2R agonists on ASM growth and promitogenic signaling. Pretreatment of healthy and asthmatic human ASM cells with TAS2R agonists resulted in a dose-dependent inhibition of ASM proliferation. The antimitogenic effect of TAS2R ligands was not dependent on activation of protein kinase A, protein kinase C, or high/intermediate-conductance calcium-activated K(+) channels. Immunoblot analyses revealed that TAS2R agonists inhibit growth factor-activated protein kinase B phosphorylation without affecting the availability of phosphatidylinositol 3,4,5-trisphosphate, suggesting TAS2R agonists block signaling downstream of phosphatidylinositol 3-kinase. Furthermore, the antimitogenic effect of TAS2R agonists involved inhibition of induced transcription factors (activator protein-1, signal transducer and activator of transcription-3, E2 factor, nuclear factor of activated T cells) and inhibition of expression of multiple cell cycle regulatory genes, suggesting a direct inhibition of cell cycle progression. Collectively, these findings establish the antimitogenic effect of TAS2R agonists and identify a novel class of receptors and signaling pathways that can be targeted to reduce or prevent airway remodeling as well as

Based on the structure of the superpotent 5-HT2A agonist 2-(4-bromo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine, which consists of a ring-substituted phenethylamine skeleton modified with an N-benzyl group, we designed and synthesized a small library of constrained analogues to identify the optimal arrangement of the pharmacophoric elements of the ligand. Structures consisted of diversely substituted tetrahydroisoquinolines, piperidines, and one benzazepine. Based on the structure of (S,S)-9b, which showed the highest affinity of the series, we propose an optimal binding conformation. (S,S)-9b also displayed 124-fold selectivity for the 5-HT2A over the 5-HT2Creceptor, making it the most selective 5-HT2A receptoragonist ligand currently known. PMID:23336049

Although endogenous estrogen is known to offer cardiac and vascular protection, the involvement of estrogen receptors in mediating the protective effect of estrogen on hypertension-induced cardiovascular and renal injury is not fully explained. We aimed to investigate the effects of estrogen receptor (ER) agonists on oxidative injury, cardiovascular and renal functions of rats with renovascular hypertension (RVH). Female Sprague-Dawley rats were randomly divided as control and RVH groups, and RVH groups had either ovariectomy (OVX) or sham-OVX. Sham-OVX-RVH and OVX-RVH groups received either ERβ agonist diarylpropiolnitrile (1 mg/kg/day) or ERα agonist propyl pyrazole triol (1 mg/kg/day) for 6 weeks starting at the third week following the surgery. At the end of the 9(th) week, systolic blood pressures were recorded, cardiac functions were determined, and the contraction/relaxation responses of aortic rings were obtained. Serum creatinine levels, tissue malondialdehyde, glutathione, superoxide dismutase, catalase levels, and myeloperoxidase activity in heart and kidney samples were analyzed, and Na(+), K(+)-ATPase activity was measured in kidney samples. In both sham-OVX and OVX rats, both agonists reduced blood pressure and reversed the impaired contractile performance of the heart, while ERβ agonist improved renal functions in both the OVX and non-OVX rats. Both agonists reduced neutrophil infiltration, lipid peroxidation, and elevated antioxidant levels in the heart, but a more ERβ-mediated protective effect was observed in the kidney. Our data suggest that activation of ERβ might play a role in preserving the function of the stenotic kidney and delaying the progression of renal injury, while both receptors mediate similar cardioprotective effects. PMID:27399230

The P2Y12 receptor (P2Y12R), one of eight members of the P2YR family expressed in humans, has been identified as one of the most prominent clinical drug targets for inhibition of platelet aggregation. Consequently, extensive mutagenesis and modeling studies of the P2Y12R have revealed many aspects of agonist/antagonist binding1-4. However, the details of agonist and antagonist recognition and function at the P2Y12R remain poorly understood at the molecular level. Here, we report the structures of the human P2Y12R in complex with a full agonist 2-methylthio-adenosine-5′-diphosphate (2MeSADP, a close analogue of endogenous agonist ADP) at 2.5 Å resolution, and the corresponding ATP derivative 2-methylthio-adenosine-5′-triphosphate (2MeSATP) at 3.1 Å resolution. Analysis of these structures, together with the structure of the P2Y12R with antagonist ethyl 6-(4-((benzylsulfonyl)carbamoyl)piperidin-1-yl)-5-cyano-2-methylnicotinate (AZD1283)5, reveals dramatic conformational changes between nucleotide and non-nucleotide ligand complexes in the extracellular regions, providing the first insight into a different ligand binding landscape in the δ-group of class A G protein-coupled receptors (GPCRs). Agonist and non-nucleotide antagonist adopt different orientations in the P2Y12R, with only partially overlapped binding pockets. The agonist-bound P2Y12R structure answers long-standing ambiguities surrounding P2Y12R-agonist recognition, and reveals interactions with several residues that had not been reported to be involved in agonist binding. As a first example of a GPCR where agonist access to the binding pocket requires large scale rearrangements in the highly malleable extracellular region, the structural studies therefore will provide invaluable insight into the pharmacology and mechanisms of action of agonists and different classes of antagonists for the P2Y12R and potentially for other closely related P2YRs. PMID:24784220

Relapse to cocaine dependence, even after extended abstinence, involves a number of liability factors including impulsivity (predisposition toward rapid, unplanned reactions to stimuli without regard to negative consequences) and cue reactivity (sensitivity to cues associated with cocaine-taking which can promote cocaine-seeking). These factors have been mechanistically linked to serotonin (5-hydroxytryptamine, 5-HT) signaling through the 5-HT(2A) receptor (5-HT(2A)R) and 5-HT(2C)R; either a selective 5-HT(2A)R antagonist or a 5-HT(2C)R agonist suppresses impulsivity and cocaine-seeking in preclinical models. We conducted proof-of-concept analyses to evaluate whether a combination of 5-HT(2A)R antagonist plus 5-HT(2C)R agonist would have synergistic effects over these liability factors for relapse as measured in a 1-choice serial reaction time task and cocaine self-administration/reinstatement assay. Combined administration of a dose of the selective 5-HT(2A)R antagonist M100907 plus the 5-HT(2C)R agonist WAY163909, each ineffective alone, synergistically suppressed cocaine-induced hyperactivity, inherent and cocaine-evoked impulsive action, as well as cue- and cocaine-primed reinstatement of cocaine-seeking behavior. The identification of synergism between a 5-HT(2A)R antagonist plus a 5-HT(2C)R agonist to attenuate these factors important in relapse indicates the promise of a bifunctional ligand as an anti-addiction pharmacotherapeutic, setting the stage to develop new ligands with improved efficacy, potency, selectivity, and in vivo profiles over the individual molecules. PMID:23336050

Aiming to design partial agonists for a G-protein-coupled receptor based on dynamic ligand binding, we synthesized three different series of bipharmacophoric ligands composed of the orthosteric building blocks iperoxo and 1 linked to allosteric modulators (BQCA-derived compounds, BQCAd; TBPB-derived compound, TBPBd). Their interactions were studied with the human muscarinic acetylcholine M1-receptor (hM1) with respect to receptor binding and Gq-protein signaling. Results demonstrate that iperoxo/BQCAd (2, 3) and 1/BQCAd hybrids (4) act as M1 partial agonists, whereas 1/TBPBd hybrids (5) did not activate M1-receptors. Among the iperoxo/BQCAd-hybrids, spacer length in conjunction with the pattern of substitution tuned efficacy. Most interestingly, a model of dynamic ligand binding revealed that the spacer length of 2a and 3a controlled the probability of switch between the inactive purely allosteric and the active bitopic orthosteric/allosteric binding pose. In summary, dynamic ligand binding can be exploited in M1 receptors to design partial agonists with graded efficacy. PMID:25478907

Due to their high affinity and specificity, aptamers have been widely used as effective inhibitors in clinical applications. However, the ability to activate protein function through aptamer-protein interaction has not been well-elucidated. To investigate their potential as target-specific agonists, we used SELEX to generate aptamers to the insulin receptor (IR) and identified an agonistic aptamer named IR-A48 that specifically binds to IR, but not to IGF-1 receptor. Despite its capacity to stimulate IR autophosphorylation, similar to insulin, we found that IR-A48 not only binds to an allosteric site distinct from the insulin binding site, but also preferentially induces Y1150 phosphorylation in the IR kinase domain. Moreover, Y1150-biased phosphorylation induced by IR-A48 selectively activates specific signaling pathways downstream of IR. In contrast to insulin-mediated activation of IR, IR-A48 binding has little effect on the MAPK pathway and proliferation of cancer cells. Instead, AKT S473 phosphorylation is highly stimulated by IR-A48, resulting in increased glucose uptake both in vitro and in vivo. Here, we present IR-A48 as a biased agonist able to selectively induce the metabolic activity of IR through allosteric binding. Furthermore, our study also suggests that aptamers can be a promising tool for developing artificial biased agonists to targeted receptors. PMID:26245346

Some forms of depression appear to be more related to the glutamatergic system. G-coupled protein receptor 39 (GPR39) is the metabotropic zinc receptor, which may be involved in the pathophysiology of depression and in the antidepressant response. Its deficiency abolishes the antidepressant response, which means that GPR39 is required to obtain a therapeutic effect in depression. This raises the possibility that agonists of the zinc receptor may have a role in antidepressant treatment. To explore this possibility we investigated animal behaviour in the forced swim test, the tail suspension test (to assess antidepressant-like properties), the light/dark test and the elevated plus maze test (to assess anxiolytic-like properties), following acute administration of a GPR39 agonist (TC G-1008). We found an antidepressant response (as measured by the forced swim test but not by the tail suspension test) in mice following the GPR39 agonist treatment. Additionally, we observed the opposite results in the light/dark box (decreased overall distance; increased time spent in the lit compartment; decreased time spent in the dark compartment; increased freezing time) and elevated plus maze (no significant changes), which may be a consequence of the sedative effect of TC G-1008. We also found hippocampal GPR39 and brain-derived neurotrophic factor (BDNF) up-regulation following administration of the GPR39 agonist, which may be undiscovered so far as a possible novel agent in the treatment of mood disorders. PMID:27235821

Due to their high affinity and specificity, aptamers have been widely used as effective inhibitors in clinical applications. However, the ability to activate protein function through aptamer-protein interaction has not been well-elucidated. To investigate their potential as target-specific agonists, we used SELEX to generate aptamers to the insulin receptor (IR) and identified an agonistic aptamer named IR-A48 that specifically binds to IR, but not to IGF-1 receptor. Despite its capacity to stimulate IR autophosphorylation, similar to insulin, we found that IR-A48 not only binds to an allosteric site distinct from the insulin binding site, but also preferentially induces Y1150 phosphorylation in the IR kinase domain. Moreover, Y1150-biased phosphorylation induced by IR-A48 selectively activates specific signaling pathways downstream of IR. In contrast to insulin-mediated activation of IR, IR-A48 binding has little effect on the MAPK pathway and proliferation of cancer cells. Instead, AKT S473 phosphorylation is highly stimulated by IR-A48, resulting in increased glucose uptake both in vitro and in vivo. Here, we present IR-A48 as a biased agonist able to selectively induce the metabolic activity of IR through allosteric binding. Furthermore, our study also suggests that aptamers can be a promising tool for developing artificial biased agonists to targeted receptors. PMID:26245346

A series of 2,2-dimethyl-3,3-diphenyl-propanamides as novel glucocorticoid receptor modulators is reported. SAR exploration led to the identification of 4-hydroxyphenyl propanamide derivatives displaying good agonist activity in GR-mediated transrepression assays and reduced agonist activity in GR-mediated transactivation assays. Compounds 17 and 30 showed anti-inflammatory activity comparable to prednisolone in the rat carrageenan-induced paw edema model, with markedly decreased side effects with regard to increases in blood glucose and expression of hepatic tyrosine aminotransferase. A hypothetical binding mode accounting for the induction of the functional activity by a 4-hydroxyl group is proposed. PMID:21073190

The gastrointestinal hormone motilin has been known about for >40 years, but after identification of its receptor and subsequent development of new tools and methods, a reappraisal of its actions is required. Firstly, it is important to note that motilin and ghrelin receptors are members of the same family (similar genomic organization, gastrointestinal distribution and abilities to stimulate gastrointestinal motility), yet each fails to recognize the ligand of the other; and whereas ghrelin and ghrelin receptors are widespread outside the gastrointestinal tract, motilin and its receptors are largely restricted to the gastrointestinal tract. Secondly, although some studies suggest motilin has activity in rodents, most do not, and receptor pseudogenes exist in rodents. Thirdly, motilin preferentially operates by facilitating enteric cholinergic activity rather than directly contracting the muscle, despite the relatively high expression of receptor immunoreactivity in muscle. This activity is ligand-dependent, with short-lasting actions of motilin contrasting with longer-lasting actions of the non-selective and selective motilin receptoragonists erythromycin and GSK962040. Finally, the use of erythromycin (also an antibiotic drug) to treat patients requiring acceleration of gastric emptying has led to concerns over safety and potential exacerbation of antibiotic resistance. Replacement motilin receptoragonists derived from erythromycin (motilides) have been unsuccessful. New, non-motilide, small molecule receptoragonists, designed to minimize self-desensitization, are now entering clinical trials for treating patients undergoing enteral feeding or with diabetic gastroparesis. Thus, for the translational pharmacologist, the study of motilin illustrates the need to avoid overreliance on artificial systems, on structural information and on animal studies. LINKED ARTICLES This article is part of a themed section on Neuropeptides. To view the other articles in this

Opioid receptors (ORs) are widely distributed in the brain, the spinal cord, and the digestive tract and play an important role in nociception. All known ORs are G-protein-coupled receptors (GPCRs) of family A. Another well-known member of this family, rhodopsin, is activated by light through the cis/trans isomerization of a covalently bound chromophore, retinal. We now show how an OR can be combined with a synthetic azobenzene photoswitch to gain light sensitivity. Our work extends the reach of photopharmacology and outlines a general strategy for converting Family A GPCRs, which account for the majority of drug targets, into photoreceptors. PMID:24519993

Site-specific deamination of five adenosine residues in the pre-mRNA of the serotonin 2Creceptor, 5HT2CR, alters the amino acid sequence of the encoded protein. Such RNA editing can produce 32 mRNA variants, encoding 24 protein isoforms that vary in biochemical and pharmacological properties. Because serotonin functions in the regulation of mood and behaviour, modulation of serotonin signalling by RNA editing may be relevant to such psychiatric disorders as anxiety and depression. Several recent human studies have reported changes in 5HT2CR editing in schizophrenia, major depression or suicide, but results are variable and not conclusive. Rodent studies have begun to examine effects of drug treatments and stress. Understanding the importance of 5HT2CR editing in mood and behaviour will be assisted by experiments designed to analyse multiple strains of mice, in different behavioural tests, with optimal evaluation of the time course of molecular changes. PMID:16769676

[11C]Cimbi-36 was recently developed as a selective serotonin 2A (5-HT2A) receptoragonist radioligand for positron emission tomography (PET) brain imaging. Such an agonist PET radioligand may provide a novel, and more functional, measure of the serotonergic system and agonist binding is more likely than antagonist binding to reflect 5-HT levels in vivo. Here, we show data from a first-in-human clinical trial with [11C]Cimbi-36. In 29 healthy volunteers, we found high brain uptake and distribution according to 5-HT2A receptors with [11C]Cimbi-36 PET. The two-tissue compartment model using arterial input measurements provided the most optimal quantification of cerebral [11C]Cimbi-36 binding. Reference tissue modeling was feasible as it induced a negative but predictable bias in [11C]Cimbi-36 PET outcome measures. In five subjects, pretreatment with the 5-HT2A receptor antagonist ketanserin before a second PET scan significantly decreased [11C]Cimbi-36 binding in all cortical regions with no effects in cerebellum. These results confirm that [11C]Cimbi-36 binding is selective for 5-HT2A receptors in the cerebral cortex and that cerebellum is an appropriate reference tissue for quantification of 5-HT2A receptors in the human brain. Thus, we here describe [11C]Cimbi-36 as the first agonist PET radioligand to successfully image and quantify 5-HT2A receptors in the human brain. PMID:24780897

The melanocortin-1 receptor (MC1R) is a key regulator of mammalian pigmentation. Melanism in the grey squirrel is associated with an eight amino acid deletion in the mutant melanocortin-1 receptor with 24 base pair deletion (MC1RΔ24) variant. We demonstrate that the MC1RΔ24 exhibits a higher basal activity than the wildtype MC1R (MC1R-wt). We demonstrate that agouti signalling protein (ASIP) is an inverse agonist to the MC1R-wt but is an agonist to the MC1RΔ24. We conclude that the deletion in the MC1RΔ24 leads to a receptor with a high basal activity which is further activated by ASIP. This is the first report of ASIP acting as an agonist to MC1R. PMID:24879893

The N-Methyl D-Aspartic acid (NMDA) receptors (NMDAR) are key tetrameric ionotropic glutamate receptors that transduce glutamatergic signals throughout the central nervous system (CNS) and spinal cord. Although NMDARs are diverse in their subunit composition, subcellular localization, and biophysical and pharmacological properties, their activation always requires the binding of a co-agonist that has long been thought to be glycine. However, intense research over the last decade has challenged this classical model by showing that another amino acid, d-serine, is the preferential co-agonist for a subset of synaptic NMDARs in many areas of the adult brain. Nowadays, a totally new picture of glutamatergic synapses at work is emerging where both glycine and d-serine are involved in a complex interplay to regulate NMDAR functions in the CNS following time and space constraints. The purpose of this review was to highlight the particular role of each co-agonist in modulating NMDAR-dependent activities in healthy and diseased brains. We have herein integrated our most advanced knowledge of how glycine and d-serine may orchestrate synapse dynamics and drive neuronal network activity in a time- and synapse-specific manner and how changes in synaptic availability of these amino acids may contribute to cognitive impairments such as those associated with healthy aging, epilepsy, and schizophrenia. The N-Methyl D-Aspartic acid (NMDA) subtype of glutamate receptors are central to many physiological functions and are linked to brain disorders. Their functions require glutamate and a co-agonist d-serine or glycine. After years of intense research and controversy on the identity of the amino acid that serves as the right co-agonist, we are just entering a new era of consensus where glycine and d-serine are teaming up to regulate the function of different subsets of NMDA receptors and at different synapses during different time windows of brain development. PMID:26088787

The concept of “functional selectivity” or “biased signaling” suggests that a ligand can have distinct efficacies with regard to different signaling pathways. We have investigated the question of whether biased signaling may be related to distinct agonist-induced conformational changes in receptors using the β2-adrenergic receptor (β2AR) and its two endogenous ligands epinephrine and norepinephrine as a model system. Agonist-induced conformational changes were determined in a fluorescently tagged β2AR FRET sensor. In this β2AR sensor, norepinephrine caused signals that amounted to only ≈50% of those induced by epinephrine and the standard “full” agonist isoproterenol. Furthermore, norepinephrine-induced changes in the β2AR FRET sensor were slower than those induced by epinephrine (rate constants, 47 versus 128 ms). A similar partial β2AR activation signal was revealed for the synthetic agonists fenoterol and terbutaline. However, norepinephrine was almost as efficient as epinephrine (and isoproterenol) in causing activation of Gs and adenylyl cyclase. In contrast, fenoterol was quite efficient in triggering β-arrestin2 recruitment to the cell surface and its interaction with β2AR, as well as internalization of the receptors, whereas norepinephrine caused partial and slow changes in these assays. We conclude that partial agonism of norepinephrine at the β2AR is related to the induction of a different active conformation and that this conformation is efficient in signaling to Gs and less efficient in signaling to β-arrestin2. These observations extend the concept of biased signaling to the endogenous agonists of the β2AR and link it to distinct conformational changes in the receptor. PMID:20837485

Animal studies have established that drugs activating the serotonin 2A (5-HT2A) receptor can enhance learning and memory in a variety of classical and operant conditioning tasks. Unfortunately, long-term agonism typically results in receptor downregulation, which can negate such nootropic effects. Conversely, chronic antagonism can act to increase receptor density, an adaptation which, in principle, should enhance cognition in a manner similar to acute agonism. In this study, we questioned whether chronic treatment with the 5-HT2A receptor antagonist, SR 46349B, a drug known to increase 5-HT2A receptor density in vivo, would improve cognitive performance in normal mice. To address this question, we administered SR 46349B to mice for 4 days following initial training on a simple rule-based reward acquisition task. We subsequently tested their recall of this task and, finally, their ability to adapt to a reversal in reward contingency (reversal learning). For comparison, two additional groups were treated with the 5-HT2A/2Creceptoragonist, DOI, which downregulates the 5-HT2A receptor. SR 46349B improved retention of the previously-learned task but did not affect reversal learning. Subjects treated with SR 46349B also completed trials faster and with greater motor efficiency than vehicle- or DOI-treated subjects. We hypothesize that long-term drug treatments resulting in 5-HT2A receptor up-regulation may be useful in enhancing recall of learned behaviors and, thus, may have potential for treating cognitive impairment associated with neurodegenerative disorders. PMID:23587729

The present study was undertaken to investigate the role of the hypothalamic tripeptide L-prolyl-L-leucyl-glycinamide (PLG) and its conformationally constrained analog 3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA) in modulating agonist binding to human dopamine (DA) receptor subtypes using human neuroblastoma SH-SY5Y cells stably transfected with respective cDNAs. Both PLG and PAOPA enhanced agonist [3H]N-propylnorapomorphine (NPA) and [3H]quinpirole binding in a dose-dependent manner to the DA D2L,D2S, and D4 receptors. However, agonist binding to the D1 and D3 receptors and antagonist binding to the D2L receptors by PLG were not significantly affected. Scatchard analysis of [3H]NPA binding to membranes in the presence of PLG revealed a significant increase in affinity of the agonist binding sites for the D2L, D2S, and D4 receptors. Analysis of agonist/antagonist competition curves revealed that PLG and PAOPA increased the population and affinity of the high-affinity form of the D2L receptor and attenuated guanosine 5'-(beta,gamma-imido)-triphosphate-induced inhibition of high-affinity agonist binding sites for the DA D2L receptor. Furthermore, direct NPA binding with D2L cell membranes pretreated with suramin, a compound that can uncouple receptor/G protein complexes, and incubated with and without DA showed that both PLG and PAOPA had only increased agonist binding in membranes pretreated with both suramin and DA, suggesting that PLG requires the D2L receptor/G protein complex to increase agonist binding. These results suggest that PLG possibly modulates DA D2S, D2L, and D4 receptors in an allosteric manner and that the coupling of D2 receptors to the G protein is essential for this modulation to occur. PMID:16126839

Behavioral effects of a nonpeptidic NOP (nociceptin/orphanin FQ Peptide) receptoragonist, Ro 64-6198, have not been studied in primate species. The aim of the study was to verify the receptor mechanism underlying the behavioral effects of Ro 64-6198 and to systematically compare behavioral effects of Ro 64-6198 with those of a mu-opioid receptoragonist, alfentanil, in monkeys. Both Ro 64-6198 (0.001-0.06 mg/kg, s.c.) and alfentanil (0.001-0.06 mg/kg, s.c.) produced antinociception against an acute noxious stimulus (50 degrees C water) and capsaicin-induced allodynia. An NOP receptor antagonist, J-113397 (0.01-0.1 mg/kg, s.c.), dose-dependently produced rightward shifts of the dose-response curve of Ro 64-6198-induced antinociception. The apparent pA(2) value of J-113397 was 8.0. Antagonist studies using J-113397 and naltrexone revealed that Ro 64-6198 produced NOP receptor-mediated antinociception independent of mu-opioid receptors. In addition, alfentanil dose-dependently produced respiratory depression and itch/scratching responses, but antinociceptive doses of Ro 64-6198 did not produce such effects. More important, Ro 64-6198 did not produce reinforcing effects comparable with those of alfentanil, cocaine, or methohexital under self-administration procedures in monkeys. These results provide the first functional evidence that the activation of NOP receptors produces antinociception without reinforcing effects in primates. Non-peptidic NOP receptoragonists may have therapeutic value as novel analgesics without abuse liability in humans. PMID:19279568

We have previously reported that, in the rat dorsolateral septal nucleus (DLSN), metabotropic glutamate receptor (met-GluR) agonists evoked a slow depolarization accompanied by an increase in membrane conductance and burst firing. We have speculated that the burst firing elicited by met-GluR agonists may be due to activation or enhancement of a non-specific cation current, which exists in some DLSN neurons. Now we report that a slow afterdepolarization (sADP) mediated by a non-specific cation current was potentiated by both 1S,3R-ACPD and quisqualate. In addition, met-GluR agonists unmask a sADP in DLSN neurons which did not show a sADP under control conditions. Our data suggest that a non-specific cation current can be potentiated by activation of the met-GluR.

New inverse agonists of the ghrelin receptor (ghrelinR) were obtained through high-throughput screening and subsequent structural modification of 2-aminoalkyl nicotinamide derivatives. The key structural feature to improve in vitro activity was the introduction of a diazabicyclo ring at the 5-position of the pyridine ring. The final product showed potent inverse agonist activity and, despite its low brain permeability, reduced food intake in both normal and obese mice. These results implied that peripheral ghrelinR activity is important for appetite control and that a peripheral ghrelinR inverse agonist could be an anti-obesity drug with reduced risk of central nervous system (CNS)-related side effects. PMID:25981690

Treatment of pain with morphine and its congeners in sickle cell anemia is suboptimal, warranting the need for analgesics devoid of side effects, addiction and tolerance liability. Small-molecule nociceptin opioid receptor ligands show analgesic efficacy in acute and chronic pain models. We show that AT-200, a high affinity nociceptin opioid receptoragonist with low efficacy at the mu opioid receptor, ameliorated chronic and hypoxia/reoxygenation-induced mechanical, thermal and deep tissue/musculoskeletal hyperalgesia in HbSS-BERK sickle mice. The antinociceptive effect of AT-200 was antagonized by SB-612111, a nociceptin opioid receptor antagonist, but not naloxone, a non-selective mu opioid receptor antagonist. Daily 7-day treatment with AT-200 did not develop tolerance and showed a sustained anti-nociceptive effect, which improved over time and led to reduced plasma serum amyloid protein, neuropeptides, inflammatory cytokines and mast cell activation in the periphery. These data suggest that AT-200 ameliorates pain in sickle mice via the nociceptin opioid receptor by reducing inflammation and mast cell activation without causing tolerance. Thus, nociceptin opioid receptoragonists are promising drugs for treating pain in sickle cell anemia. PMID:26294734

Treatment of pain with morphine and its congeners in sickle cell anemia is suboptimal, warranting the need for analgesics devoid of side effects, addiction and tolerance liability. Small-molecule nociceptin opioid receptor ligands show analgesic efficacy in acute and chronic pain models. We show that AT-200, a high affinity nociceptin opioid receptoragonist with low efficacy at the mu opioid receptor, ameliorated chronic and hypoxia/reoxygenation-induced mechanical, thermal and deep tissue/musculoskeletal hyperalgesia in HbSS-BERK sickle mice. The antinociceptive effect of AT-200 was antagonized by SB-612111, a nociceptin opioid receptor antagonist, but not naloxone, a non-selective mu opioid receptor antagonist. Daily 7-day treatment with AT-200 did not develop tolerance and showed a sustained anti-nociceptive effect, which improved over time and led to reduced plasma serum amyloid protein, neuropeptides, inflammatory cytokines and mast cell activation in the periphery. These data suggest that AT-200 ameliorates pain in sickle mice via the nociceptin opioid receptor by reducing inflammation and mast cell activation without causing tolerance. Thus, nociceptin opioid receptoragonists are promising drugs for treating pain in sickle cell anemia. PMID:26294734

Human tissues express cannabinoid CB(1) and CB(2) receptors that can be activated by endogenously released 'endocannabinoids' or exogenously administered compounds in a manner that reduces the symptoms or opposes the underlying causes of several disorders in need of effective therapy. Three medicines that activate cannabinoid CB(1)/CB(2) receptors are now in the clinic: Cesamet (nabilone), Marinol (dronabinol; Δ(9)-tetrahydrocannabinol (Δ(9)-THC)) and Sativex (Δ(9)-THC with cannabidiol). These can be prescribed for the amelioration of chemotherapy-induced nausea and vomiting (Cesamet and Marinol), stimulation of appetite (Marinol) and symptomatic relief of cancer pain and/or management of neuropathic pain and spasticity in adults with multiple sclerosis (Sativex). This review mentions several possible additional therapeutic targets for cannabinoid receptoragonists. These include other kinds of pain, epilepsy, anxiety, depression, Parkinson's and Huntington's diseases, amyotrophic lateral sclerosis, stroke, cancer, drug dependence, glaucoma, autoimmune uveitis, osteoporosis, sepsis, and hepatic, renal, intestinal and cardiovascular disorders. It also describes potential strategies for improving the efficacy and/or benefit-to-risk ratio of these agonists in the clinic. These are strategies that involve (i) targeting cannabinoid receptors located outside the blood-brain barrier, (ii) targeting cannabinoid receptors expressed by a particular tissue, (iii) targeting upregulated cannabinoid receptors, (iv) selectively targeting cannabinoid CB(2) receptors, and/or (v) adjunctive 'multi-targeting'. PMID:23108552

There is mounting evidence that increased brain serotonin during exercise is associated with the onset of CNS-mediated fatigue. Serotonin receptor sensitivity is likely to be an important determinant of this fatigue. Alterations in brain serotonin receptor sensitivity were examined in Wistar rats throughout 6 weeks of endurance training, running on a treadmill four times a week with two exercise tests per week to exhaustion. Receptor sensitivity was determined indirectly as the reduction in exercise time in response to a dose of a serotonin (1A) agonist, m-chlorophenylpiperazine (m-CPP). The two groups of controls were used to examine (i) the effect of the injection per se on exercise performance and (ii) changes in serotonin receptor sensitivity associated with maturation. In the test group, undrugged exercise performance significantly improved by 47% after 6 weeks of training (4518 +/- 729 to 6640 +/- 903 s, P=0.01). Drugged exercise performance also increased significantly from week 1 to week 6 (306 +/- 69-712 +/- 192 s, P = 0.04). Control group results indicated that the dose of m-CPP alone caused fatigue during exercise tests and that maturation was not responsible for any decrease in receptor sensitivity. Improved resistance to the fatiguing effects of the serotonin agonist suggests desensitization of central serotonin receptors, probably the 5-HT1A receptors. Endurance training appears to stimulate an adaptive response to the fatiguing effects of increased brain serotonin, which may enhance endurance exercise performance. PMID:11167306

Human tissues express cannabinoid CB1 and CB2 receptors that can be activated by endogenously released ‘endocannabinoids’ or exogenously administered compounds in a manner that reduces the symptoms or opposes the underlying causes of several disorders in need of effective therapy. Three medicines that activate cannabinoid CB1/CB2 receptors are now in the clinic: Cesamet (nabilone), Marinol (dronabinol; Δ9-tetrahydrocannabinol (Δ9-THC)) and Sativex (Δ9-THC with cannabidiol). These can be prescribed for the amelioration of chemotherapy-induced nausea and vomiting (Cesamet and Marinol), stimulation of appetite (Marinol) and symptomatic relief of cancer pain and/or management of neuropathic pain and spasticity in adults with multiple sclerosis (Sativex). This review mentions several possible additional therapeutic targets for cannabinoid receptoragonists. These include other kinds of pain, epilepsy, anxiety, depression, Parkinson's and Huntington's diseases, amyotrophic lateral sclerosis, stroke, cancer, drug dependence, glaucoma, autoimmune uveitis, osteoporosis, sepsis, and hepatic, renal, intestinal and cardiovascular disorders. It also describes potential strategies for improving the efficacy and/or benefit-to-risk ratio of these agonists in the clinic. These are strategies that involve (i) targeting cannabinoid receptors located outside the blood-brain barrier, (ii) targeting cannabinoid receptors expressed by a particular tissue, (iii) targeting upregulated cannabinoid receptors, (iv) selectively targeting cannabinoid CB2 receptors, and/or (v) adjunctive ‘multi-targeting’. PMID:23108552

Citrus fruit and citrus fruit products, like grapefruit, lemon and marmalade were shown to contain aryl hydrocarbon receptor (AhR) agonists, as detected with the DR CALUX bioassay. This is of interest regarding the role of the Ah-receptor pathway in the adverse effects of dioxins, PCBs and other aromatic hydrocarbons. So far it is unclear which compounds in citrus fruit are responsible for the AhR-mediated activity and whether regular exposure to these compounds can cause effects comparable to, e.g. dioxins. The present study aimed at developing a method for identifying unknown Ah-receptoragonists in citrus products based on bioassay directed analysis, using marmalade as a first target. Following extraction with hexane and purification on an aluminium oxide-column, the extract was fractionated by HPLC using a C-18 semi-preparative column. Fractions were extracted, solvent-exchanged into dimethylsulfoxide and subsequently tested with DR CALUX. Extracts were shown to contain primarily coumarins, furocoumarins (FCs) and polymethoxyflavones (PMFs). Identification of fractions most active in the bioassay via LC/MS revealed that bergapten (an FC) is the most important Ah-receptoragonist in marmalade. The approach and method developed resulted in the successful identification of the bioactive component. However, potential pitfalls of the procedure will be discussed. PMID:18486664

The central melanocortin (MC) system has been widely studied for its effects on food intake and sexual behavior. However, the MC system, and more specifically the MC4 receptor (MC4R), also interacts with neurochemical systems that regulate socioemotional behaviors, including oxytocin (OT) and dopamine. In monogamous prairie voles, OT and dopamine interact to promote partner preference formation, a laboratory measure of an enduring social bond between mates. Here we investigated the effects of MC receptor activation on partner preference formation in prairie voles, as well as the interaction between the MC and OT systems during this process. Peripheral administration of the brain penetrant MC3/4R receptor peptide agonist, Melanotan II (MTII), and the highly selective, small-molecule MC4R agonist, Pf-446687, enhanced partner preference formation in the prairie vole, but not in the non-monogamous meadow vole. MTII-induced partner preferences were enduring, as they were present 1 week after drug manipulation. The prosocial effects of MCR agonists may be mediated, in part, through modulation of OT, as coadministration of an OT receptor antagonist prevented MTII-induced partner preferences. MTII also selectively activated hypothalamic OT neurons and potentiated central OT release. As OT has been shown to enhance some aspects of social cognition in humans, our data suggest that the MC4R may be a viable therapeutic target for enhancing social function in psychiatric disorders, including autism spectrum disorders and schizophrenia, potentially through activation of the OT system. PMID:25652247

Intra-abdominal hypertension (IAH) is accompanied by high morbidity and mortality in surgical departments and ICUs. However, its specific pathophysiology is unclear. IAH not only leads to intra-abdominal tissue damage but also causes dysfunction in distal organs, such as the brain. In this study, we explore the protective effects of melanocortin 4 receptoragonists in IAH-induced brain injury. The IAH rat models were induced by hemorrhagic shock/resuscitation (with the mean arterial pressure (MAP) maintained at 30 mm Hg for 90 min followed by the reinfusion of the withdrawn blood with lactated Ringer's solution). Then, air was injected into the peritoneal cavity of the rats to maintain an intra-abdominal pressure of 20 mm Hg for 4 h. The effects of the melanocortin 4 receptoragonist RO27-3225 in alleviating the rats' IAH brain injuries were observed, which indicated that RO27-3225 could reduce brain edema, the expressions of the IL-1β and TNF-α inflammatory cytokines, the blood-brain barrier's permeability and the aquaporin4 (AQP4) and matrix metalloproteinase 9 (MMP9) levels. Moreover, the nicotinic acetylcholine receptor antagonist chlorisondamine and the selective melanocortin 4 receptor antagonist HS024 can negate the protective effects of the RO27-3225. The MC4R agonist can effectively reduce the intracerebral proinflammatory cytokine gene expression and alleviate the brain injury caused by blood-brain barrier damage following IAH. PMID:25616531

The central melanocortin (MC) system has been widely studied for its effects on food intake and sexual behavior. However, the MC system, and more specifically the MC4 receptor (MC4R), also interacts with neurochemical systems that regulate socioemotional behaviors, including oxytocin (OT) and dopamine. In monogamous prairie voles, OT and dopamine interact to promote partner preference formation, a laboratory measure of an enduring social bond between mates. Here we investigated the effects of MC receptor activation on partner preference formation in prairie voles, as well as the interaction between the MC and OT systems during this process. Peripheral administration of the brain penetrant MC3/4R receptor peptide agonist, Melanotan II (MTII), and the highly selective, small-molecule MC4R agonist, Pf-446687, enhanced partner preference formation in the prairie vole, but not in the non-monogamous meadow vole. MTII-induced partner preferences were enduring, as they were present 1 week after drug manipulation. The prosocial effects of MCR agonists may be mediated, in part, through modulation of OT, as coadministration of an OT receptor antagonist prevented MTII-induced partner preferences. MTII also selectively activated hypothalamic OT neurons and potentiated central OT release. As OT has been shown to enhance some aspects of social cognition in humans, our data suggest that the MC4R may be a viable therapeutic target for enhancing social function in psychiatric disorders, including autism spectrum disorders and schizophrenia, potentially through activation of the OT system. PMID:25652247

The formyl peptide receptor 2 (FPR2) is a remarkably versatile transmembrane protein belonging to the G-protein coupled receptor (GPCR) family. FPR2 is activated by an array of ligands, which include structurally unrelated lipids and peptide/proteins agonists, resulting in different intracellular responses in a ligand-specific fashion. In addition to the anti-inflammatory lipid, lipoxin A4, several other endogenous agonists also bind FPR2, including serum amyloid A, glucocorticoid-induced annexin 1, urokinase and its receptor, suggesting that the activation of FPR2 may result in potent pro- or anti-inflammatory responses. Other endogenous ligands, also present in biological samples, include resolvins, amyloidogenic proteins, such as beta amyloid (Aβ)-42 and prion protein (Prp)106–126, the neuroprotective peptide, humanin, antibacterial peptides, annexin 1-derived peptides, chemokine variants, the neuropeptides, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP)-27, and mitochondrial peptides. Upon activation, intracellular domains of FPR2 mediate signaling to G-proteins, which trigger several agonist-dependent signal transduction pathways, including activation of phospholipase C (PLC), protein kinase C (PKC) isoforms, the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway, the mitogen-activated protein kinase (MAPK) pathway, p38MAPK, as well as the phosphorylation of cytosolic tyrosine kinases, tyrosine kinase receptor transactivation, phosphorylation and nuclear translocation of regulatory transcriptional factors, release of calcium and production of oxidants. FPR2 is an attractive therapeutic target, because of its involvement in a range of normal physiological processes and pathological diseases. Here, we review and discuss the most significant findings on the intracellular pathways and on the cross-communication between FPR2 and tyrosine kinase receptors triggered by different FPR2 agonists. PMID

We measured the functional agonist potencies of dopamine agonists including antiparkinson drugs, and functional antagonist potencies of antipsychotics at human dopamine D(2) and D(3) receptors. In vitro pharmacological assessment included inhibition of forskolin-stimulated cAMP accumulation and the reversal of dopamine-induced inhibition in clonal Chinese hamster ovary cells expressing low and high densities of human dopamine D(2L) and D(2S) receptors (hD(2L)-Low, hD(2L)-High, hD(2S)-Low and hD(2S)-High, respectively) and human dopamine D(3) Ser-9 and D(3) Gly-9 receptors (hD(3)-Ser-9 and hD(3)-Gly-9, respectively). Cabergoline, bromocriptine, pergolide, (±)-7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT), talipexole, pramipexole, R-(+)-trans-3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-olhydrochloride (PD128907) and ropinirole behaved as dopamine D(2) and D(3) receptor full agonists and showed higher potencies in hD(2L)-High and hD(2S)-High compared to hD(2L)-Low and hD(2S)-Low. In hD(3)-Ser-9 and hD(3)-Gly-9 compared to hD(2L)-Low and hD(2S)-Low, dopamine, ropinirole, PD128907, and pramipexole potencies were clearly higher; talipexole and 7-OH-DPAT showed slightly higher potencies; pergolide showed slightly lower potency; and, cabergoline and bromocriptine potencies were lower. Aripiprazole acted as an antagonist in hD(2L)-Low; a low intrinsic activity partial agonist in hD(2S)-Low; a moderate partial agonist in hD(3)-Ser-9 and hD(3)-Gly-9; a robust partial agonist in hD(2L)-High; and a full agonist in hD(2S)-High. Amisulpride, sulpiride and perphenazine behaved as preferential antagonists; and chlorpromazine and asenapine behaved as modest preferential antagonists; whereas fluphenazine, haloperidol, and blonanserin behaved as non-preferential antagonists in hD(2S)-Low and hD(2S)-High compared to hD(3)-Ser-9 and hD(3)-Gly-9. These findings may help to elucidate the basis of therapeutic benefit observed with these drugs, with

We have studied the effects of groups I, II, and III metabotropic glutamate receptor (mGluR) agonists and antagonists on pyloric activity in the stomatogastric ganglion (STG) of the Caribbean spiny lobster Panulirus argus. We have found that agonists for all three groups of mGluRs modify the pyloric output. The group I agonist, l-quisqualic acid (l-QA), activated the pyloric central pattern generator (CPG). When the pyloric rhythm was partially suppressed by sucrose-block of input fibers in the stomatogastric nerve (stn), l-QA accelerated the rhythmic activity. In addition, the number of spike discharges was increased in pyloric motoneurons: pyloric (PY), and lateral pyloric (LP). In completely blocked preparations, a slow pyloric rhythm was initiated by l-QA. Groups II and III agonists exerted an inhibitory effect on pyloric activity. The group II agonist, (2S,1'S,2'S)-2-(Carboxycyclopropyl)glycine (L-CCG-I), decreased both the frequency of the pyloric rhythm and the number of spike discharges in the motoneurons: ventricular dilator (VD), PY, and LP. The effects of L-CCG-I were dose-dependent. The group III agonist, l-(+)-2-Amino-4-phosphonobutyric acid (l-AP4), slightly decreased the frequency of the pyloric rhythm and suppressed spike discharges in the VD neuron. All effects of mGluR agonists were reversible. The effect of l-QA was blocked by the broad spectrum mGluR antagonist (S)-Methyl-4-carboxyphenylglycine (MCPG). The inhibitory effect of L-CCG-I was prevented by MCPG and by the group II/III mGluR antagonist (RS)-alpha-Methyl-4-phosphonophenylglycine (MPPG), and was partially blocked by the group II mGluR antagonist (RS)-1-amino-5-phosphonoindan-1-carboxylic acid (APICA). The inhibitory effect of l-AP4 was blocked by MPPG and partially blocked by APICA. PMID:16256086

Glucocorticoids (GCs) are widely used as anti-inflammatory drugs. Our previous study demonstrated that several GCs such as cortisol and dexamethasone (Dex) were frequently detected in effluents collected from Japanese sewage treatment plants (STPs) in 2012. In this study, we used the GC-Responsive Chemical-Activated LUciferase gene eXpression (GR-CALUX) assay to elucidate GC receptor (GR) agonistic activities of ten pure synthetic GCs and selected STP effluents in Japan for assessment of the risks associated with the presence of GR agonists. The tested GCs demonstrated dose-dependent agonistic effects in the GR-CALUX assay and their EC50 values were calculated for estimation of relative potencies (REPs) compared to Dex. The GR agonistic potency was in the rank of: clobetasol propionate > clobetasone butyrate > betamethasone 17-valerate > difluprednate > betamethasone 17,21-dipropionate > Dex > betamethasone > 6α-methylprednisolone > prednisolone > cortisol. The GR agonistic activity in STP effluents as measured in Dex-equivalent (Dex-EQ) activities ranged from < 3.0-78 ng L(-1) (median: 29 ng L(-1), n = 50). To evaluate the contribution of the target GCs, theoretical Dex-EQs were calculated by multiplying the concentrations of each GC by its respective REP. Our calculation of Dex-EQ contribution for individual GR agonists indicated that the well-known GCs cortisol and Dex should not be given priority for subsequent in vivo testing, monitoring and removal experiments, but rather the highly potent synthetic GCs clobetasol propionate and betamethasone 17-valerate (REP = 28 and 3.1) as well as other unidentified compounds are important GR agonists in STP effluents in Japan. PMID:25965047

The tetrahydronaphthalene-benzoxazine glucocorticoid receptor (GR) partial agonist 4b was optimized to produce potent full agonists of GR. Aromatic ring substitution of the tetrahydronaphthalene leads to weak GR antagonists. Discovery of an "agonist trigger" substituent on the saturated ring of the tetrahydronaphthalene leads to increased potency and efficacious GR agonism. These compounds are efficacy selective in an NFkB GR agonist assay (representing transrepression effects) over an MMTV GR agonist assay (representing transactivation effects). 52 and 60 have NFkB pIC(50) = 8.92 (105%) and 8.69 (92%) and MMTV pEC(50) = 8.20 (47%) and 7.75 (39%), respectively. The impact of the trigger substituent on agonism is modeled within GR and discussed. 36, 52, and 60 have anti-inflammatory activity in a mouse model of inflammation after topical dosing with 52 and 60, having an effect similar to that of dexamethasone. The original lead was discovered by a manual agreement docking method, and automation of this method is also described. PMID:16821781

Peroxisome proliferator-activated receptor (PPAR) agonists are used for treating hyperglycemia and type 2 diabetes. However, the mechanism of action of these agonists is still under investigation. The lipid droplet-associated proteins FSP27/CIDEC and LSDP5, regulated directly by PPARγ and PPARα, are associated with hepatic steatosis and insulin sensitivity. Here, we evaluated the expression levels of FSP27/CIDEC and LSDP5 and the regulation of these proteins by consumption of a high-fat diet (HFD) or administration of PPAR agonists. Mice with diet-induced obesity were treated with the PPARγ or PPARα agonist, pioglitazone or fenofibrate, respectively. Liver tissues from db/db diabetic mice and human were also collected. Interestingly, FSP27/CIEDC was expressed in mouse and human livers and was upregulated in obese C57BL/6J mice. Fenofibrate treatment decreased hepatic triglyceride (TG) content and FSP27/CIDEC protein expression in mice fed an HFD diet. In mice, LSDP5 was not detected, even in the context of insulin resistance or treatment with PPAR agonists. However, LSDP5 was highly expressed in humans, with elevated expression observed in the fatty liver. We concluded that fenofibrate greatly decreased hepatic TG content and FSP27/CIDEC protein expression in mice fed an HFD, suggesting a potential regulatory role for fenofibrate in the amelioration of hepatic steatosis. PMID:26770990

Agonists of the nuclear receptor PPARγ are therapeutically used to combat hyperglycaemia associated with the metabolic syndrome and type 2 diabetes. In spite of being effective in normalization of blood glucose levels, the currently used PPARγ agonists from the thiazolidinedione type have serious side effects, making the discovery of novel ligands highly relevant. Natural products have proven historically to be a promising pool of structures for drug discovery, and a significant research effort has recently been undertaken to explore the PPARγ-activating potential of a wide range of natural products originating from traditionally used medicinal plants or dietary sources. The majority of identified compounds are selective PPARγ modulators (SPPARMs), transactivating the expression of PPARγ-dependent reporter genes as partial agonists. Those natural PPARγ ligands have different binding modes to the receptor in comparison to the full thiazolidinedione agonists, and on some occasions activate in addition PPARα (e.g. genistein, biochanin A, sargaquinoic acid, sargahydroquinoic acid, resveratrol, amorphastilbol) or the PPARγ-dimer partner retinoid X receptor (RXR; e.g. the neolignans magnolol and honokiol). A number of in vivo studies suggest that some of the natural product activators of PPARγ (e.g. honokiol, amorfrutin 1, amorfrutin B, amorphastilbol) improve metabolic parameters in diabetic animal models, partly with reduced side effects in comparison to full thiazolidinedione agonists. The bioactivity pattern as well as the dietary use of several of the identified active compounds and plant extracts warrants future research regarding their therapeutic potential and the possibility to modulate PPARγ activation by dietary interventions or food supplements. PMID:25083916

Dopamine D3 receptor (D3 R) is considered as a potential target for the treatment of nervous system disorders, such as Parkinson's disease. Current research interests primarily focus on the discovery and design of potent D3 agonists. In this work, we selected 40 D3 R agonists as the research system. Comparative molecular field analysis (CoMFA) of three-dimensional quantitative structure-activity relationship (3D-QSAR), structure-selectivity relationship (3D-QSSR), and molecular docking was performed on D3 receptoragonists to obtain the details at atomic level. The results indicated that both the CoMFA model (r(2) = 0.982, q(2) = 0.503, rpred2 = 0.893, SEE = 0.057, F = 166.308) for structure-activity and (r(2) = 0.876, q(2) = 0.436, rpred2 = 0.828, F = 52.645) for structure-selectivity have good predictive capabilities. Furthermore, docking studies on three compounds binding to D3 receptor were performed to analyze the binding modes and interactions. The results elucidate that agonists formed hydrogen bond and hydrophobic interactions with key residues. Finally, we designed six molecules under the guidance of 3D-QSAR/QSSR models. The activity and selectivity of designed molecules have been improved, and ADMET properties demonstrate they have low probability of hepatotoxicity (<0.5). These results from 3D-QSAR/QSSR and docking studies have great significance for designing novel dopamine D3 selective agonists in the future. PMID:26851125

AIM: To evaluate the inflammasome activation and the effect of peroxisome proliferator-activated receptors (PPAR)-δ agonist treatment in nonalcoholic fatty liver disease (NAFLD) models. METHODS: Male C57BL/6J mice were classified according to control or high fat diet (HFD) with or without PPAR-δ agonist (GW) over period of 12 wk [control, HFD, HFD + lipopolysaccharide (LPS), HFD + LPS + GW group]. HepG2 cells were exposed to palmitic acid (PA) and/or LPS in the absence or presence of GW. RESULTS: HFD caused glucose intolerance and hepatic steatosis. In mice fed an HFD with LPS, caspase-1 and interleukin (IL)-1β in the liver were significantly increased. Treatment with GW ameliorated the steatosis and inhibited overexpression of pro-inflammatory cytokines. In HepG2 cells, PA and LPS treatment markedly increased mRNA of several nucleotide-binding and oligomerization domain-like receptor family members (NLRP3, NLRP6, and NLRP10), caspase-1 and IL-1β. PA and LPS also exaggerated reactive oxygen species production. All of the above effects of PA and LPS were reduced by GW. GW also enhanced the phosphorylation of AMPK-α. CONCLUSION: PPAR-δ agonist reduces fatty acid-induced inflammation and steatosis by suppressing inflammasome activation. Targeting the inflammasome by the PPAR-δ agonist may have therapeutic implication for NAFLD. PMID:26668503

The aryl hydrocarbon receptor (AHR) is well-known for its role in mediating the toxic and adaptive responses to xenobiotic compounds. Recent studies also indicate that AHR ligands are endogenously produced and may be essential for normal development. Previously, we showed that the endogenous enzyme, aspartate aminotransferase (AST), generates the AHR proagonist, indole-3-pyruvic acid (I3P), by deamination of its substrate L-tryptophan. We hypothesized that other enzymatic pathways capable of producing I3P may generate AHR agonists in vivo. We now demonstrate that the enzyme d-amino acid oxidase (DAAO) catalyzes the production of AHR agonists through the enzymatic conversion of D-tryptophan to I3P. Moreover, we provide evidence that the nonenzymatic oxidation and condensation of I3P is a critical step in the generation of receptoragonists by DAAO and AST. Products of this process include two novel agonists, 1,3-di(1H-indol-3-yl)propan-2-one and 1-(1H-indol-3-yl)-3-(3H-indol-3-ylidene) propan-2-one [characterized in the accompanying paper, Chowdhury et al. ( 2009 ) Chem. Res. Toxicol. , DOI: 10.1021/tx9000418 ], both of which can potently activate the AHR at concentrations in the nanomolar range. These results show that endogenous AHR activity can be modulated by I3P production from amino acid precursors through multiple enzymatic pathways, including those catalyzed by DAAO and AST. PMID:19860415

Cannabinoid receptor 2 agonists and inverse agonists are emerging as new therapeutic options for a spectrum of autoimmune-related disease. Of particular interest, is the ability of CB2 ligands to regulate microglia function in neurodegenerative diseases and traumatic brain injury. We have previously reported the receptor affinity of 3′,5′-dichloro-2,6-dihydroxy-biphenyl-4-yl)-phenyl-methanone (SMM-189) and the characterization of the beneficial effects of SMM-189 in the mouse model of mild traumatic brain injury. Herein, we report the further characterization of SMM-189 as a potent and selective CB2 inverse agonist, which acts as a noncompetitive inhibitor of CP 55,940. The ability of SMM-189 to regulate microglial activation, in terms of chemokine expression and cell morphology, has been determined. Finally, we have determined that SMM-189 possesses acceptable biopharmaceutical properties indicating that the triaryl class of CB2 inverse agonists are viable compounds for continued preclinical development for the treatment of neurodegenerative disorders and traumatic brain injury. PMID:26196013

Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A2AR-D2R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D2R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A2AR agonists, but also A2AR antagonists, decrease the affinity and intrinsic efficacy of D2R agonists and the affinity of D2R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A2AR-D2R heteromers as heterotetramers, constituted by A2AR and D2R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain. PMID:26100888

Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A2AR-D2R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D2R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A2AR agonists, but also A2AR antagonists, decrease the affinity and intrinsic efficacy of D2R agonists and the affinity of D2R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A2AR-D2R heteromers as heterotetramers, constituted by A2AR and D2R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain. PMID:26100888

A thyroid hormone receptor beta subtype-selective thyromimetic 5 was found to be efficacious in both mouse and monkey hair growth models after topical applications. It penetrates the skin according to the test in human cadaver skin mounted onto Franz diffusion chambers. The serum drug level of 5 is below the limit of quantification during tests in the bald stump-tailed macaques (Macaca arctoides). It is tested negative in the 3T3 neutral red uptake (NRU) phototoxicity test, indicating a low risk for causing photo-irritation. It is also rapidly metabolized according to the PK data, thus the systemic exposure is limited. PMID:19900809

Selexipag [2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide] is an orally available prostacyclin (PGI(2)) receptor (IP receptor) agonist that is chemically distinct from PGI(2) and is in clinical development for the treatment of pulmonary arterial hypertension. Selexipag is highly selective for the human IP receptor in vitro, whereas analogs of PGI(2) can activate prostanoid receptors other than the IP receptor. The goal of this study was to determine the impact of selectivity for the IP receptor on gastric function by measuring 1) contraction of rat gastric fundus ex vivo and 2) the rates of gastric emptying and intestinal transport in response to selexipag in comparison with other PGI(2) analogs. The rat gastric fundus expresses mRNA encoding multiple prostanoid receptors to different levels: prostaglandin E receptor 1 (EP(1)) > prostaglandin E receptor 3 (EP(3)), IP receptor > prostaglandin D(2) receptor 1, thromboxane receptor. Selexipag and metabolite {4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}acetic acid (ACT-333679) did not contract gastric fundus at concentrations up to 10(-3) M. In contrast, the PGI(2) analogs iloprost and beraprost evoked concentration-dependent contraction of gastric fundus. Contraction to treprostinil was observed at high concentration (10(-4) M). Contraction to all PGI(2) analogs was mediated via activation of EP(3) receptors, although EP(1) receptors also contributed to the contraction of gastric fundus to iloprost and beraprost. Antagonism of IP receptors did not affect responses. Oral selexipag did not significantly alter gastric function in vivo, as measured by rates of stomach emptying and intestinal transport, whereas beraprost slowed gastrointestinal transport. The high functional selectivity of selexipag and ACT-333679 for the IP receptor precludes a stimulatory action on gastric smooth muscle and may help minimize gastric side effects such as nausea and vomiting. PMID:20660124

Dopamine receptor mechanisms are believed to play a role in the reinforcing effects of cocaine and other drugs of abuse. The lack of receptor-selective agonists has made it difficult to determine the role of the individual dopamine receptors in mediating these reinforcing effects. In this study, rhesus monkeys with a history of intravenous cocaine self-administration were tested for the reinforcing effects of several D(3)-preferring agonists, a D(2)-preferring agonist, and a D(4) agonist. The D(2)-preferring agonist did not maintain responding in any monkeys, and the D(4) agonist was self-administered at low rates, just above those maintained by saline, in one monkey. The D(3)-preferring agonists were self-administered by approximately half of the animals, although at lower rates than cocaine. These results indicate that the apparent limited reinforcing effectiveness of D(2)-like agonists requires activity at D(3) receptors. Previous data from this laboratory and others also suggest that these drugs may not serve as reinforcers directly; the behavior may be maintained by response-contingent delivery of stimuli previously paired with cocaine. The ability of drug-related stimuli to maintain responding apparently differs among monkeys and other organisms, and may be related to individual differences in drug-taking behavior in humans. PMID:22785383

Neuromedin U (NMU) are bioactive peptides with a common C-terminal heptapeptide sequence (FLFRPRN-amide, 1a) among mammals, which is responsible for receptor activation, namely NMU receptor types 1 (NMUR1) and 2 (NMUR2). Among the various physiological actions of NMU, the anorexigenic effect has recently attracted attention in drug discovery efforts for treating obesity. Although several structure-activity relationship (SAR) studies have been reported, receptor-selective small peptide agonists have yet to be disclosed. Herein a SAR study of 1a-derived peptide derivatives is described. We initially screened both human NMUR1- and NMUR2-selective peptides in calcium-mobilization assays with cells transiently expressing receptors. Then we performed a precise assay with a stable expression system of receptors and consequently discovered hexapeptides 8d and 6b possessing selective agonist activity toward each respective receptor. Hexapeptide 6b, which selectively activates NMUR2 without significant NMUR1 activation, should aid in the development of anorexigenic drugs as well as advance NMU-related endocrinological research. PMID:24999562

Many antidepressant drugs interact with σ receptors and accumulating evidence suggests that these proteins mediate antidepressant-like effects in animals and humans. σ Receptors are localized in brain regions affected in depression, further strengthening the hypothesis that they represent logical drug development targets. In this study, two novel σ receptoragonists (UMB23, UMB82) were evaluated for antidepressant-like activity in mice. First, radioligand binding studies confirmed that the novel compounds had preferential affinity for σ receptors. Second, the forced swim test, a well established animal model for screening potential antidepressant drugs, showed that both compounds dose-dependently reduced immobility time. The σ receptor antagonist BD1047 attenuated the antidepressant-like effects of UMB23 and UMB82. Third, locomotor activity suggested that the effects of UMB23 and UMB82 in the forced swim test were not due to non-specific motor activating effects. Together, the data provide further evidence that σ receptoragonists represent a possible new class of antidepressant medication. PMID:17376658

Molecular-dynamics simulations with metadynamics enhanced sampling reveal three distinct binding sites for arginine vasopressin (AVP) within its V2 -receptor (V2 R). Two of these, the vestibule and intermediate sites, block (antagonize) the receptor, and the third is the orthosteric activation (agonist) site. The contacts found for the orthosteric site satisfy all the requirements deduced from mutagenesis experiments. Metadynamics simulations for V2 R and its V1a R-analog give an excellent correlation with experimental binding free energies by assuming that the most stable binding site in the simulations corresponds to the experimental binding free energy in each case. The resulting three-site mechanism separates agonists from antagonists and explains subtype selectivity. PMID:27184628

Clonidine and guanfacine are alpha-2 receptoragonists that decrease sympathetic outflow from the central nervous system. Posttraumatic stress disorder (PTSD) is an anxiety disorder that is theorized to be related to a hyperactive sympathetic nervous system. Currently, the only US Food and Drug Administration (FDA)-approved medications for PTSD are the selective serotonin reuptake inhibitors (SSRIs) sertraline and paroxetine. Sometimes use of the SSRIs may not lead to full remission and symptoms of hyperarousal often persist. This article specifically reviews the literature on alpha-2 receptoragonist use for the treatment of PTSD and concludes that while the evidence base is limited, these agents might be considered useful when SSRIs fail to treat symptoms of agitation and hyperarousal in patients with PTSD. PMID:26322115

Clonidine and guanfacine are alpha-2 receptoragonists that decrease sympathetic outflow from the central nervous system. Posttraumatic stress disorder (PTSD) is an anxiety disorder that is theorized to be related to a hyperactive sympathetic nervous system. Currently, the only US Food and Drug Administration (FDA)-approved medications for PTSD are the selective serotonin reuptake inhibitors (SSRIs) sertraline and paroxetine. Sometimes use of the SSRIs may not lead to full remission and symptoms of hyperarousal often persist. This article specifically reviews the literature on alpha-2 receptoragonist use for the treatment of PTSD and concludes that while the evidence base is limited, these agents might be considered useful when SSRIs fail to treat symptoms of agitation and hyperarousal in patients with PTSD. PMID:26322115

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors and comprise three different isoforms namely PPARα, PPARγ, and PPARβ/δ with PPARβ/δ being the predominant subtype in human keratinocytes. After binding with specific ligands, PPARs regulate gene expression, cell growth and differentiation, apoptosis, inflammatory responses, and tumorogenesis. PPARs also modulate a wide variety of skin functions including keratinocyte proliferation, epidermal barrier formation, wound healing, melanocyte proliferation, and sebum production. Recent studies have shown the importance of PPARs in the pathogenesis of many dermatological disorders. Clinical trials have suggested possible role of PPAR agonists in the management of various dermatoses ranging from acne vulgaris, psoriasis, hirsutism, and lipodystrophy to cutaneous malignancies including melanoma. This article is intended to be a primer for dermatologists in their understanding of clinical relevance of PPARs and PPAR agonists in dermatology therapeutics. PMID:25986745

The active constituents of Cannabis sativa have been used for centuries as recreational drugs and medicinal agents. Today, marijuana is the most prevalent drug of abuse in the United States and, conversely, therapeutic use of marijuana constituents are gaining mainstream clinical and political acceptance. Given the documented contributions of endocannabinoid signaling to a range of physiological systems, including cognitive function, and the control of eating behaviors, it is unsurprising that cannabinoid receptoragonists and antagonists are showing significant clinical potential. In addition to the neuroactive effects of cannabinoids, an emerging body of data suggests that both endogenous and exogenous cannabinoids are potently immunoactive. The central premise of this review article is that the immunological effects of cannabinoids should be considered in the context of each prescribing decision. We present evidence that the immunological effects of cannabinoid receptoragonists and antagonists are highly relevant to the spectrum of disorders for which cannabinoid therapeutics are currently offered. PMID:20219697

The cannabinoid receptor 2 (CB2R) has been linked with the regulation of inflammation, and selective receptor activation has been proposed as a target for the treatment of a range of inflammatory diseases such as atherosclerosis and arthritis. In order to identify selective CB2R agonists with appropriate physicochemical and ADME properties for future evaluation in vivo, we first performed a ligand-based virtual screen. Subsequent medicinal chemistry optimisation studies led to the identification of a new class of selective CB2R agonists. Several examples showed high levels of activity (EC50

Several melatonin receptorsagonists (ramelteon, prolonged-release melatonin, agomelatine and tasimelteon) have recently become available for the treatment of insomnia, depression and circadian rhythms sleep-wake disorders. The efficacy and safety profiles of these compounds in the treatment of the indicated disorders are reviewed. Accumulating evidence indicates that sleep-wake disorders and co-existing medical conditions are mutually exacerbating. This understanding has now been incorporated into the new Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-5). Therefore, when evaluating the risk/benefit ratio of sleep drugs, it is pertinent to also evaluate their effects on wake and comorbid condition. Beneficial effects of melatonin receptoragonists on comorbid neurological, psychiatric, cardiovascular and metabolic symptomatology beyond sleep regulation are also described. The review underlines the beneficial value of enhancing physiological sleep in comorbid conditions. PMID:25207602

The various physiological actions of the neurohormone melatonin are mediated mainly by two G-protein-coupled MT(1) and MT(2) receptors. The melatoninergic drugs on the market, ramelteon and agomelatine, as well as the most advanced drug candidates under clinical evaluation, tasimelteon and PD-6735, are high-affinity nonselective MT(1) and MT(2) agonists. However, exploring the exact physiological role of the MT(1) and MT(2) melatonin receptors requires subtype selective MT(1) and MT(2) ligands. This review covers novel melatoninergic agonists and antagonists published since 2010, focusing on high-affinity and subtype selective agents. Additionally, compounds not mentioned in the previous review articles and ligands selective for the MT(3) binding site are included. PMID:22680635

T0901317 is a potent non-steroidal synthetic liver X receptor (LXR) agonist. T0901317 blocked androgenic stimulation of the proliferation of androgen-dependent LNCaP 104-S cells and androgenic suppression of the proliferation of androgen-independent LNCaP 104-R2 cells, inhibited the transcriptional activation of an androgen-dependent reporter gene by androgen, and suppressed gene and protein expression of prostate specific antigen (PSA), a target gene of androgen receptor (AR) without affecting gene and protein expression of AR. T0901317 also inhibited binding of a radiolabeled androgen to AR, but inhibition was much weaker compared to the effect of the antiandrogens, bicalutamide and hydroxyflutamide. The LXR agonist T0901317, therefore, acts as an antiandrogen in human prostate cancer cells.

In rats, the discriminative stimulus effects of direct- and indirect-acting dopamine receptoragonists are mediated by multiple dopamine receptor subtypes and the relative contribution of dopamine D2 and D3 receptors to these effects varies as a function of feeding condition. In these studies, free-fed and food-restricted mice were trained to discriminate 10.0 mg/kg cocaine using a two-lever discrimination procedure in which responding was maintained by food. Both groups of mice acquired the discrimination; however, free-fed mice responded at lower rates than food-restricted mice. Dopamine D3 receptoragonists, pramipexole and quinpirole, increased cocaine-appropriate responding (>85%) in food-restricted, but not in free-fed mice. The dopamine D2 receptoragonist, sumanirole, and the nonselective dopamine receptoragonist, apomorphine, failed to increase cocaine-appropriate responding in either group. Free-fed mice were more sensitive than food-restricted mice to the rate-decreasing effects of dopamine receptoragonists and these effects could not be overcome by increasing the magnitude of reinforcement. Because feeding condition did not alter quinpirole-induced hypothermia, it is unlikely that differences in the discriminative stimulus or rate-decreasing effects of dopamine D2-like receptoragonists were due to differences in the pharmacokinetic properties of the drugs. Although these results suggest that the discriminative stimulus effects of cocaine are mediated by both dopamine D2 and D3 receptors in food-restricted mice, the increased sensitivity of free-fed mice to the rate-decreasing effects of dopamine D2-like receptoragonists limited conclusions about the impact of feeding conditions on the relative contribution of dopamine D2 and D3 receptors to the discriminative stimulus effects of cocaine. PMID:24561049

Most commonly originating from breast malignancies, metastatic bone cancer causes bone destruction and severe pain. Although novel chemotherapeutic agents have increased life expectancy, patients are experiencing higher incidences of fracture, pain, and drug-induced side effects; furthermore, recent findings suggest that patients are severely undertreated for their cancer pain. Strong analgesics, namely opiates, are first-line therapy in alleviating cancer-related pain despite the severe side effects, including enhanced bone destruction with sustained administration. Bone resorption is primarily treated with bisphosphonates, which are associated with highly undesirable side effects, including nephrotoxicity and osteonecrosis of the jaw. In contrast, cannabinoid receptor 2 (CB2) receptor-specific agonists have been shown to reduce bone loss and stimulate bone formation in a model of osteoporosis. CB2 agonists produce analgesia in both inflammatory and neuropathic pain models. Notably, mixed CB1/CB2 agonists also demonstrate a reduction in ErbB2-driven breast cancer progression. Here we demonstrate for the first time that CB2 agonists reduce breast cancer–induced bone pain, bone loss, and breast cancer proliferation via cytokine/chemokine suppression. Studies used the spontaneously-occurring murine mammary cell line (66.1) implanted into the femur intramedullary space; measurements of spontaneous pain, bone loss, and cancer proliferation were made. The systemic administration of a CB2 agonist, JWH015, for 7 days significantly attenuated bone remodeling, assuaged spontaneous pain, and decreased primary tumor burden. CB2-mediated effects in vivo were reversed by concurrent treatment with a CB2 antagonist/inverse agonist but not with a CB1 antagonist/inverse agonist. In vitro, JWH015 reduced cancer cell proliferation and inflammatory mediators that have been shown to promote pain, bone loss, and proliferation. Taken together, these results suggest CB2 agonists as a

The melanocortins (alpha-melanocyte-stimulating hormone and adrenocorticotropin) act on epidermal melanocytes to increase melanogenesis, the eumelanin/pheomelanin ratio and dendricity. These actions are mediated by the heptahelical melanocortin 1 receptor (MC1R), positively coupled to adenylyl cyclase. Gain-of-function mouse Mc1r alleles are associated with a dark, eumelanic coat. Conversely, loss-of-function variants, or overexpression of agouti, a natural melanocortin antagonist, yield yellow, pheomelanic furs. In humans, loss-of-function MC1R variants are associated with fair skin, poor tanning, propensity to freckle and increased skin cancer risk. Therefore, MC1R is a key regulator of mammalian pigmentation. Several observations such as induction of constitutive pigmentation in amelanotic mouse melanoma cells following expression of MC1R indicate that the receptor might display agonist-independent activity. We report a systematic and comparative study of MC1R and Mc1r constitutive activity. We show that expression of MC1R in heterologous systems leads to an agonist-independent increase in cyclic adenosine monophophate (cAMP). Basal signalling is a function of receptor expression and is two to fourfold higher for MC1R than for Mc1r. Moreover, it is observed in human melanoma cells over-expressing the MC1R. Constitutive signalling is abolished or reduced by point mutations of MC1R impairing the response to agonists, and is only doubled by the Lys94Glu mutation, mimicking the constitutively active mouse E(so-3J) allele. Stable or transient expression of wild-type MC1R, but not of loss-of-function mutants, potently stimulates forskolin activation of adenylyl cyclase, a common feature of constitutively active Gs-coupled receptors. Therefore, human MC1R displays a strong agonist-independent constitutive activity. PMID:15250941

The farnesoid X receptor (FXR) may play a crucial role in a number of metabolic diseases and, as such, could potentially serve as a target for the development of therapeutics as a treatment for those diseases. Previous work has described GW4064 as an FXR agonist with an interesting activity profile. This manuscript will describe the synthesis of novel analogs of GW4064 and the activity profile of those analogs. PMID:25499883

A series of N-benzylated-5-methoxytryptamine analogues was prepared and investigated, with special emphasis on substituents in the meta position of the benzyl group. A parallel series of several N-benzylated analogues of 2,5-dimethoxy-4-iodophenethylamine (2C-I) also was included for comparison of the two major templates (i.e., tryptamine and phenethylamine). A broad affinity screen at serotonin receptors showed that most of the compounds had the highest affinity at the 5-HT2 family receptors. Substitution at the para position of the benzyl group resulted in reduced affinity, whereas substitution in either the ortho or the meta position enhanced affinity. In general, introduction of a large lipophilic group improved affinity, whereas functional activity often followed the opposite trend. Tests of the compounds for functional activity utilized intracellular Ca2+ mobilization. Function was measured at the human 5-HT2A, 5-HT2B, and 5-HT2Creceptors, as well as at the rat 5-HT2A and 5-HT2Creceptors. There was no general correlation between affinity and function. Several of the tryptamine congeners were very potent functionally (EC50 values from 7.6 to 63 nM), but most were partial agonists. Tests in the mouse head twitch assay revealed that many of the compounds induced the head twitch and that there was a significant correlation between this behavior and functional potency at the rat 5-HT2A receptor. PMID:25547199

Mitogen-activated protein kinase phosphatase 1 (MKP-1) expression is induced by inflammatory factors, and it is an endogenous suppressor of inflammatory response. MKP-1 expression is increased by PDE4 inhibitor rolipram suggesting that it is regulated by cAMP-enhancing compounds. Therefore, we investigated the effect of β2-receptoragonists on MKP-1 expression and inflammatory response. We found that β2-receptoragonists salbutamol and terbutaline, as well as 8-Br-cAMP, increased MKP-1 expression. Salbutamol and terbutaline also inhibited p38 MAPK phosphorylation and TNF production in J774 mouse macrophages. Interestingly, salbutamol suppressed carrageenan-induced paw inflammation in wild-type mice, but the effect was attenuated in MKP-1(-/-) mice. In conclusion, these data show that β2-receptoragonists increase MKP-1 expression, which seems to mediate, at least partly, the observed anti-inflammatory effects of β2-receptoragonists. PMID:26849227

Mitogen-activated protein kinase phosphatase 1 (MKP-1) expression is induced by inflammatory factors, and it is an endogenous suppressor of inflammatory response. MKP-1 expression is increased by PDE4 inhibitor rolipram suggesting that it is regulated by cAMP-enhancing compounds. Therefore, we investigated the effect of β2-receptoragonists on MKP-1 expression and inflammatory response. We found that β2-receptoragonists salbutamol and terbutaline, as well as 8-Br-cAMP, increased MKP-1 expression. Salbutamol and terbutaline also inhibited p38 MAPK phosphorylation and TNF production in J774 mouse macrophages. Interestingly, salbutamol suppressed carrageenan-induced paw inflammation in wild-type mice, but the effect was attenuated in MKP-1(-/-) mice. In conclusion, these data show that β2-receptoragonists increase MKP-1 expression, which seems to mediate, at least partly, the observed anti-inflammatory effects of β2-receptoragonists. PMID:26849227

The case of a 22-year-old male Caucasian driver is presented. He was involved in a traffic collision. At the roadside, he displayed blank stare and mellow speech with a barely audible voice. A DRE found low body temperature, rigid muscle tone, normal pulse, lack of horizontal and vertical gaze nystagmus, nonconvergence of the eyes, dilated pupil size, and normal Pupillary reaction to light. A standard toxicology DUID protocol was performed on the driver's whole blood including ELISA and GC-MS drug screens with negative results. Additional drug screening was undertaken for bath salts and synthetic cannabinoid receptoragonists by LC-MS/MS by a commercial laboratory and identified the synthetic cannabinoid receptoragonist XLR-11 in the driver's blood. XLR-11 was subsequently quantified at 1.34 ng/mL. This is the first documented case involving a driver operating a motor vehicle under the influence of the synthetic cannabinoid receptoragonist XLR-11. PMID:25088081

The prevalence of type 2 diabetes is increasing at an astounding rate. Many of the agents used to treat type 2 diabetes have undesirable adverse effects of hypoglycemia and weight gain. Glucagon-like peptide-1 (GLP-1) receptoragonists represent a unique approach to the treatment of diabetes, with benefits extending outside glucose control, including positive effects on weight, blood pressure, cholesterol levels, and beta-cell function. They mimic the effects of the incretin hormone GLP-1, which is released from the intestine in response to food intake. Their effects include increasing insulin secretion, decreasing glucagon release, increasing satiety, and slowing gastric emptying. There are currently four approved GLP-1 receptoragonists in the United States: exenatide, liraglutide, albiglutide, and dulaglutide. A fifth agent, lixisenatide, is available in Europe. There are important pharmacodynamic, pharmacokinetic, and clinical differences of each agent. The most common adverse effects seen with GLP-1 therapy include nausea, vomiting, and injection-site reactions. Other warnings and precautions include pancreatitis and thyroid cell carcinomas. GLP-1 receptoragonists are an innovative and effective option to improve blood glucose control, with other potential benefits of preserving beta-cell function, weight loss, and increasing insulin sensitivity. Once-weekly formulations may also improve patient adherence. Overall, these are effective agents for patients with type 2 diabetes, who are either uncontrolled on metformin or intolerant to metformin. PMID:26213556

Almost 70% of breast cancers are estrogen receptor α (ERα) positive. Tamoxifen, a selective estrogen receptor modulator (SERM), represents the standard of care for many patients; however, 30-50% develop resistance, underlining the need for alternative therapeutics. Paradoxically, agonists at ERα such as estradiol (E2) have demonstrated clinical efficacy in patients with heavily treated breast cancer, although side effects in gynecological tissues are unacceptable. A drug that selectively mimics the actions of E2 in breast cancer therapy but minimizes estrogenic effects in other tissues is a novel, therapeutic alternative. We hypothesized that a selective human estrogen receptor partial agonist (ShERPA) at ERα would provide such an agent. Novel benzothiophene derivatives with nanomolar potency in breast cancer cell cultures were designed. Several showed partial agonist activity, with potency of 0.8-76 nM, mimicking E2 in inhibiting growth of tamoxifen-resistant breast cancer cell lines. Three ShERPAs were tested and validated in xenograft models of endocrine-independent and tamoxifen-resistant breast cancer, and in contrast to E2, ShERPAs did not cause significant uterine growth. PMID:26681208

The apelinergic system includes a series of endogenous peptides apelin, ELABELA/TODDLER and their 7-transmembrane G-protein coupled apelin receptor (APJ, AGTRL-1, APLNR). The APJ receptor is an attractive therapeutic target because of its involvement in cardiovascular diseases and potentially other disorders including liver fibrosis, obesity, diabetes, and neuroprotection. To date, pharmacological characterization of the APJ receptor has been limited due to the lack of small molecule functional agonists or antagonists. Through focused screening we identified a drug-like small molecule agonist hit 1 with a functional EC50 value of 21.5±5μM and binding affinity (Ki) of 5.2±0.5μM. Initial structure-activity studies afforded compound 22 having a 27-fold enhancement in potency and the first sub-micromolar full agonist with an EC50 value of 800±0.1nM and Ki of 1.3±0.3μM. Preliminary SAR, synthetic methodology, and in vitro pharmacological characterization indicate this scaffold will serve as a favorable starting point for further refinement of APJ potency and selectivity. PMID:27369451

Migraine headache is a highly prevalent chronic, episodic condition. The direct and indirect costs of migraine headache have a tremendous economic impact in the US. Research has shown that serotonin (5HT(1B/D)) receptoragonists reduce healthcare costs, improve health-related QOL (HR-QOL), decrease migraine disability and keep patients effective in the workplace. The purpose of this manuscript is to examine the cost effectiveness of oral 5HT(1B/D) receptoragonists for the treatment of migraine headache. In general, 5HT(1B/D) receptoragonists are associated with increases in direct healthcare costs; however, they are also associated with reductions in the indirect costs associated with migraine headache. Therefore, it appears that the relatively high acquisition cost of these medications is offset and, as a class, these medications appear to be cost effective and demonstrate net benefits from the societal perspective. Based on meta-analyses in which data on eletriptan were not available, it appears that within the class, almotriptan and rizatriptan are the most cost effective. In a prospective study comparing eletriptan with sumatriptan, it appears that the former may be more cost effective than the latter. Additional investigations are needed to further explore the application of the friction-cost approach and QALYs to cost-effectiveness analyses of this class of medication. PMID:15836007

The galanin N-terminal fragment (galanin-(1-16)) has been prepared by solid-phase synthesis and by enzymic cleavage of galanin by endoproteinase Asp-N. This peptide fragment displaced {sup 125}I-labeled galanin in receptor autoradiography experiments on rat forebrain and spinal cord and in equilibrium binding experiments from high-affinity binding sites in the ventral hippocampus with an IC50 of approximately 3 nM. In tissue slices of the same brain area, galanin-(1-16), similarly to galanin, inhibited the muscarinic agonist-stimulated breakdown of inositol phospholipids. Upon intracerebroventricular administration, galanin-(1-16) (10 micrograms/15 microliters) also inhibited the scopolamine (0.3 mg/kg, s.c.)-evoked release of acetylcholine, as studied in vivo by microdialysis. Substitution of (L-Trp2) for (D-Trp2) resulted in a 500-fold loss in affinity as compared with galanin-(1-16). It is concluded that, in the ventral hippocampus, the N-terminal galanin fragment (galanin-(1-16)) is recognized by the galanin receptors controlling acetylcholine release and muscarinic agonist-stimulated inositol phospholipid breakdown as a high-affinity agonist and that amino acid residue (Trp2) plays an important role in the receptor-ligand interactions.

Background and Purpose Rotigotine acts as a dopamine receptoragonist with high affinity for the dopamine D2, D3, D4 and D5 receptors but with a low affinity for the dopamine D1 receptor. We have investigated this further in radioligand binding and functional studies and compared the profile of rotigotine with that of other drugs used in the treatment of Parkinson's disease (PD). Experimental Approach The binding of rotigotine to human dopamine D1, D2, D3, D4 and D5 receptors was determined in radioligand binding studies using [3H]rotigotine and compared with that of standard antagonist radioligands. Functional interactions of rotigotine with human dopamine receptors was also determined. Key Results [3H]rotigotine can be used as an agonist radioligand to label all dopamine receptor subtypes and this can be important to derive agonist affinity estimates. Rotigotine maintains this high affinity in functional studies at all dopamine receptors especially D1, D2 and D3 receptors and, to a lesser extent, D4 and D5 receptors. Rotigotine, like apomorphine but unlike ropinirole and pramipexole, was a potent agonist at all dopamine receptors. Conclusions and Implications Rotigotine is a high-potency agonist at human dopamine D1, D2 and D3 receptors with a lower potency at D4 and D5 receptors. These studies differentiate rotigotine from conventional dopamine D2 agonists, used in the treatment of PD, such as ropinirole and pramipexole which lack activity at the D1 and D5 receptors, but resembles that of apomorphine which has greater efficacy in PD than other dopamine agonists but has suboptimal pharmacokinetic properties. PMID:25339241

1. This study investigated the recognition characteristics of neurokinin receptors mediating potentiation of the contractile response to field stimulation in the guinea-pig vas deferens. 2. A predominant NK1 receptor population is strongly suggested by the relative activities of the common naturally-occurring tachykinin agonists, which fall within less than one order of magnitude. This conclusion is supported by the relative activities of the synthetic NK1 selective agonists substance P methyl ester, [Glp6,L-Pro9]-SP(6-11) and delta-aminovaleryl-[L-Pro9,N-MeLeu10]- SP(7-11) (GR73632) which were 0.78, 9.3 and 120 as active as substance P, respectively. Furthermore, the NK2 selective agonist [Lys3, Gly8,-R-gamma-lactam-Leu9]-NKA(3-10) (GR64349) was active only at the highest concentrations tested (greater than 10 microM), and the NK3 selective agonist, succ-[Asp6,N-MePhe8]-SP(6-11) (senktide) was essentially inactive (10 nM-32 microM). 3. [D-Arg1,D-Pro2,D-Trp7,9,Leu11]-SP(1-11) antagonized responses to neurokinin A, neurokinin B, physalaemin, eledoisin, [Glp6,D-Pro9]-SP(6-11), GR73632 and GR64349 (apparent pKB s 5.6-6.2), but was less potent in antagonizing responses to substance P, substance P methyl ester and [Glp6,L-Pro9]-SP(6-11) (apparent pKB s less than or equal to 5.0-5.0). 4. In contrast, the recently developed NK1-selective receptor antagonist [D-Pro9[Spiro-gamma-lactam]Leu10,Trp11]-SP(1-11) (GR71251) did not produce agonist-dependent pKB estimates. Schild plot analysis indicated a competitive interaction with a single receptor population where the antagonist had an estimated overall pKB of 7.58 +/- 0.13 for the four agonists of differing subtype selectivity tested (GR73632, GR64349, substance P methyl ester and neurokinin B).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1707714

Abuse and dependence to heroin has evolved into a global epidemic as a significant clinical and societal problem with devastating consequences. Repeated exposure to heroin can induce long-lasting behavioral sensitization and withdrawal. Pharmacological activation of 5-HT2Creceptors (5-HT2CRs) suppresses psychostimulant-induced drug-seeking and behavioral sensitization. The present study examined the effect of a selective 5-HT2CR agonist lorcaserin on behavioral sensitization and naloxone-precipitated withdrawal symptoms in heroin-treated mice. Male mice received heroin (1.0 mg/kg, s.c.) twice a day for 3 days and then drug treatment was suspended for 5 days. On day 9, a challenge dose of heroin (1.0 mg/kg) was administered to examine the expression of behavioral sensitization. Lorcaserin administered during the development, withdrawal or expression stage suppressed heroin-induced behavioral sensitization on day 9. Another cohort of mice received increasing doses of heroin over a 4.5-day period. Lorcaserin, or the positive control clonidine (an α2-adrenoceptor agonist) suppressed naloxone-precipitated withdrawal symptoms in heroin-treated mice. These findings suggest that activation of 5-HT2CRs suppresses behavioral sensitization and withdrawal in heroin-treated mice. Thus, pharmacological activation of 5-HT2CRs may represent a new avenue for the treatment of heroin addiction. PMID:26375926

Three group I mGluR antagonists CPCCOEt, LY367385 and BAY36-7620, were analyzed for their effect on cell surface expression of metabotropic glutamate receptor 1a and 1b. All three antagonists inhibited glutamate-induced internalization of mGluR1a and mGluR1b. However, when added alone, either LY367385 or BAY36-7620 increased the cell surface expression of mGluR1a but not mGluR1b. Both LY367385 and BAY36-7620 displayed inverse agonist activity as judged by their ability to inhibit basal inositol phosphate accumulation in cells expressing the constitutively active mGluR1a. Interestingly, mGluR1a but not mGluR1b was constitutively internalized in HEK293 cells and both LY367385 and BAY36-7620 inhibited the constitutive internalization of this splice variant. Furthermore, coexpression of dominant negative mutant constructs of arrestin-2 [arrestin-2-(319-418)] or Eps15 [Eps15(E Delta 95-295)] increased cell surface expression of mGluR1a and blocked constitutive receptor internalization. In the presence of these dominant negative mutants, incubation of cells with LY367385 and BAY36-7620 produced no further increase in cell surface expression of mGluR1a. Taken together, these results suggest that the constitutive activity of mGluR1a triggers the internalization of the receptor through an arrestin- and clathrin-dependent pathway, and that inverse agonists increase the cell surface expression of mGluR1a by promoting an inactive form of mGluR1a, which does not undergo constitutive internalization. PMID:15140199

Background Lipid lowering agent such as agonists of peroxisome proliferator-activated receptors (PPAR) are suggested as neuroprotective agents and may protect from the sequelae of brain ischemic stroke. Although the demonstration is not clearly established in human, the underlying molecular mechanism may be of interest for future therapeutic purposes. To this end, we have used our well established rodent model of ischemia-reperfusion pre-treated or not with fenofibrate or atorvastatin and performed a differential proteomics analyses of the brain and analysed the protein markers which levels returned to “normal” following pre-treatments with PPARα agonists. Results In order to identify potential therapeutic targets positively modulated by pre-treatment with the PPARα agonists, two-dimensional gel electrophoresis proteome profiles between control, ischemia-reperfusion and pre-treated or not, were compared. The polypeptide which expression was altered following ischemia – reperfusion but whose levels remain unchanged after pre-treatment were characterized by mass spectrometry and further investigated by Western-blotting and immunohistochemistry. A series of 28 polypeptides were characterized among which the protein disulfide isomerase reduction – a protein instrumental to the unfolded protein response system - was shown to be reduced following PPARα agonists treatment while it was strongly increased in ischemia-reperfusion. Conclusions Pre-treatment with PPARα agonist or atorvastatin show potential neuroprotective effects by inhibiting the PDI overexpression in conjunction with the preservation of other neuronal markers, several of which are associated with the regulation of protein homeostasis, signal transduction and maintenance of synaptic plasticity. This proteomic study therefore suggests that neuroprotective effect of PPARα agonists supposes the preservation of the expression of several proteins essential for the maintenance of protein homeostasis

The retinoic acid receptor-related receptors (RORs) are members of the nuclear receptor (NR) superfamily of transcription factors. Several NRs are still characterized as orphan receptors since ligands have not yet been identified for these proteins. Here, we describe the identification of a synthetic RORα/RORγ ligand, SR1078. SR1078 modulates the conformation of RORγ in a biochemical assay and activates RORα and RORγ driven transcription. Furthermore, SR1078 stimulates expression of endogenous ROR target genes in HepG2 cells that express both RORα and RORγ. Pharmacokinetic studies indicate that SR1078 displays reasonable exposure following injection into mice and consistent with SR1078 functioning as a RORα/RORγ agonist, expression of two ROR target genes, glucose-6-phosphatase and fibroblast growth factor 21, were stimulated in the liver. Thus, we have identified the first synthetic RORα/γ agonist and this compound can be utilized as a chemical tool to probe the function of these receptors both in vitro and in vivo. PMID:20735016

Activation of GABA(B) receptors leads to longer inhibitory postsynaptic potentials than activation of GABA(A) receptors. Therefore GABA(B) receptors may be a target for anticonvulsant therapy. The present study examined possible effects of GABA(B) receptoragonist SKF97541 on cortical and hippocampal epileptic afterdischarges (ADs). Epileptic ADs elicited by electrical stimulation of sensorimotor cortex or dorsal hippocampus were studied in adult male Wistar rats. Stimulation series were applied 6 times with 10- or 20-min interval. Either interval was efficient for reliable elicitation of cortical ADs but stimulation at 10-min intervals did not reliably elicit hippocampal ADs, many stimulations were without effect. SKF97541 in dose 1 mg/kg significantly prolonged cortical ADs. Duration of hippocampal ADs was not significantly changed by either dose of SKF97541 in spite of a marked myorelaxant effect of the higher dose. Our present data demonstrated that neither cortical nor hippocampal ADs in adult rats were suppressed by GABA(B) receptoragonist SKF97541. Proconvulsant effect on cortical ADs indicates a different role in these two brain structures. In addition, duration of refractory period for electrically-induced ADs in these two structures in adult rats is different. PMID:24702499

The agonist radioligand (-)-N6-(125I)-p-hydroxyphenylisopropyl-adenosine (( 125I)HPIA) was used to characterize adenosine recognition sites in porcine atrial membranes. (125I)HPIA showed saturable binding to an apparently homogeneous population of sites with a maximum binding capacity of 35 +/- 3 fmol/mg of protein and an equilibrium dissociation constant of 2.5 +/- 0.4 nM. Kinetic experiments were performed to address the molecular mechanism of (125I)HPIA binding in porcine atrial membranes. (125I)HPIA apparently interacts with the cardiac adenosine receptor in a simple bimolecular reaction. A kinetically derived (125I) HPIA dissociation constant (2.4 nM) was in good agreement with that parameter measured at equilibrium. Guanyl nucleotides negatively modulated (125I)HPIA binding by increasing its rate of dissociation. This finding is consonant with the formation of a ternary complex in porcine atrial membranes, consisting of ligand, receptor, and guanyl nucleotide-binding protein. Prototypic adenosine receptoragonists and antagonists inhibited specific binding in a manner consistent with the labeling of an A1 adenosine receptor. The results of these experiments suggest that the adenosine receptor present in porcine atrial membranes, as labeled by (125I)HPIA, is of the A1 subtype.

The epilepsies are a heterogeneous collection of seizure disorders with a lifetime expectancy risk rate of 2-4%. A convergence of evidence indicates that heritable factors contribute significantly to seizure susceptibility. Genetically epilepsy-prone rodent strains have been frequently used to examine the effect of genetic factors on seizure susceptibility. The most extensively studied of these have been strains that are susceptible to sound-induced convulsions (audiogenic seizures, or AGSs). Early observations of the AGS phenomenon were made in the laboratory of Dr. Ivan Pavlov; in the course of appetite-conditioning experiments in mice, the loud bell used to signal food presentation unexpectedly produced seizures in some animals. In 1947, DBA/2 (D2) mice were found to exhibit a genetic susceptibility to AGSs stimulated by a doorbell mounted in an iron tub. Since this discovery, AGSs have been among the most intensively studied phenotypes in behavioural genetics. Although several genetic loci confer susceptibility to AGSs, the corresponding genes have not been cloned. We report that null mutant mice lacking serotonin 5-HT2Creceptors are extremely susceptible to AGSs. The onset of susceptibility is between two and three months of age, with complete penetrance in adult animals. AGS-induced immediate early gene expression indicates that AGSs are subcortical phenomena in auditory circuits. This AGS syndrome is the first produced by a known genetic defect; it provides a robust model for the examination of serotoninergic mechanisms in epilepsy. PMID:9241279

The detection and discrimination of diverse chemical structures by the vertebrate olfactory system is accomplished by the recognition of odorous ligands by their cognate receptors. In the present study we used a computational high-throughput screening strategy to discover novel high affinity agonists of an olfactory G protein-coupled receptor tuned to recognize amino acid ligands. Functional testing of the top candidates validated several agonists with potencies higher than any of the receptor’s known natural ligands. Computational modeling revealed molecular interactions involved in ligand recognition by this receptor, and further highlighted interactions that have been conserved in evolutionarily divergent amino acid receptors. Significantly, the top compounds display robust activities as odorants in vivo, and include a natural product that may be used to signal the presence of bacteria in the aquatic environment. Our virtual screening approach should be applicable to the identification of new bioactive molecules for probing the structure of chemosensory receptors and the function of chemosensory systems in vivo. PMID:19081373

A quantitative systems pharmacology model that combines in vitro/preclinical neurophysiology data, human imaging data, and patient disease information was used to blindly predict steady-state clinical efficacy of vabicaserin, a 5-HT2C full agonist, in monotherapy and, subsequently, to assess adjunctive therapy in schizophrenia. The model predicted a concentration-dependent improvement of positive and negative syndrome scales (PANSS) in schizophrenia monotherapy with vabicaserin. At the exposures of 100 and 200 mg b.i.d., the predicted improvements on PANSS in virtual patient trials were 5.12 (2.20, 8.56) and 6.37 (2.27, 10.40) (mean (95% confidence interval)), respectively, which are comparable to the observed phase IIa results. At the current clinical exposure limit of vabicaserin, the model predicted an ~9-point PANSS improvement in monotherapy, and <4-point PANSS improvement adjunctive with various antipsychotics, suggesting limited clinical benefit of vabicaserin in schizophrenia treatment. In conclusion, the updated quantitative systems pharmacology model of PANSS informed the clinical development decision of vabicaserin in schizophrenia. PMID:24759548

Previous work has shown that continuous estradiol replacement in young ovariectomized rats enhances acquisition of a delayed matching-to-position (DMP) T-maze task over that of ovariectomized controls. The mechanism by which estradiol confers this benefit has not been fully elucidated. This study examined the role of selective estrogen receptoragonists of ERα, ERβ, and GPR30 in the enhancement of spatial learning on a DMP task by comparing continuous estradiol replacement with continuous administration of PPT (an agonist of ERα), DPN (an agonist of ERβ), or G-1 (an agonist of GPR30) relative to gonadally intact and ovariectomized vehicle-treated controls. It was found that ovariectomy impaired acquisition on this task, whereas all ER selective agonists restored the rate of acquisition to that of gonadally intact controls. These data suggest that estradiol can work through any of several estrogen receptors to enhance the rate of acquisition on this task. PMID:19560466

The highly potent micro -opioid receptoragonist 14-methoxymetopon (4,5alpha-epoxy-3-hydroxy-14beta-methoxy-5beta,17-dimethylmorphinan-6-one) was prepared in tritium labelled form by a catalytic dehalogenation method resulting in a specific radioactivity of 15.9 Ci/mmol. Opioid binding characteristics of [3H]14-methoxymetopon were determined using radioligand binding assay in rat brain membranes. [3H]14-Methoxymetopon specifically labelled a single class of opioid sites with affinity in low subnanomolar range (Ki = 0.43 nm) and maximal number of binding sites of 314 fmol/mg protein. Binding of [3H]14-methoxymetopon was inhibited by ligands selective for the micro -opioid receptor with high potency, while selective kappa-opioids and delta-opioids were weaker inhibitors. 14-Methoxymetopon increased guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS) binding with an EC50 of 70.9 nm, thus, providing evidence for the agonist character of this ligand. The increase of [35S]GTPgammaS binding was inhibited by naloxone and selective micro -opioid antagonists, indicating a micro -opioid receptor-mediated action. [3H]14-Methoxymetopon is one of the few nonpeptide mu-opioid receptoragonists available in radiolabelled form up to now. Due to its high affinity and selectivity, high stability and extremely low nonspecific binding (<10%), this radioligand would be an important and useful tool in probing mu-opioid receptor mechanisms, as well as to promote a further understanding of the opioid system at the cellular and molecular level. PMID:12887410

Ractopamine (RAC) is fed to an estimated 80% of all beef, swine, and turkey raised in the United States. It promotes muscle mass development, limits fat deposition, and reduces feed consumption. However, it has several undesirable behavioral side effects in livestock, especially pigs, including restlessness, agitation, excessive oral-facial movements, and aggressive behavior. Numerous in vitro and in vivo studies suggest RAC's physiological actions begin with its stimulation of β1- and β2-adrenergic receptor-mediated signaling in skeletal muscle and adipose tissue; however, the molecular pharmacology of RAC's psychoactive effects is poorly understood. Using human cystic fibrosis transmembrane conductance regulator (hCFTR) chloride channels as a sensor for intracellular cAMP, we found that RAC and p-tyramine (TYR) produced concentration-dependent increases in chloride conductance in oocytes coexpressing hCFTR and mouse trace amine-associated receptor 1 (mTAAR1), which was completely reversed by the trace amine-associated receptor 1 (TAAR1)-selective antagonist EPPTB [N-(3-ethoxyphenyl)-4-pyrrolidin-1-yl-3-trifluoromethylbenzamide]. Oocytes coexpressing hCFTR and the human β2-adrenergic receptor showed no response to RAC or TYR. These studies demonstrate that, contrary to expectations, RAC is not an agonist of the human β2-adrenergic receptor but rather a full agonist for mTAAR1. Since TAAR1-mediated signaling can influence cardiovascular tone and behavior in several animal models, our finding that RAC is a full mTAAR1 agonist supports the idea that this novel mechanism of action influences the physiology and behavior of pigs and other species. These findings should stimulate future studies to characterize the pharmacological, physiological, and behavioral actions of RAC in humans and other species exposed to this drug. PMID:24799633

Despite the recent successes in producing orexin receptor subtype-selective antagonists, these are not commonly available, and therefore, agonist ligands are regularly used to ascribe cell and tissue responses to OX(1) or OX(2) receptors. In the current study, we have compared the native "subtype-selective" agonist, orexin-B, and its reputedly enhanced synthetic variant, Ala(11), d-Leu(15)-orexin-B, in two different recombinant cell lines. Ca2+ elevation was used as readout, and the two "selective" ligands were compared to the subtype-non-selective orexin-A, as is customary with these ligands. In transiently transfected HEK-293 cells, orexin-B showed 9-fold selectivity for the OX(2) receptor and Ala(11), d-Leu(15)-orexin-B 23-fold selectivity, when the potency ratios of ligands were compared between OX(1) and OX(2). In stable CHO-K1 cells, the corresponding values were only 2.6- and 14-fold, respectively. In addition to being low, the selectivity of the ligands was also variable, as indicated by the comparison of the two cell lines. For instance, the relative potency of Ala(11), d-Leu(15)-orexin-B at OX(2) in CHO cells was only 2.3-fold higher than its relative potency at OX(1) in HEK-293 cells; this indicates that Ala(11), d-Leu(15)-orexin-B does not show high enough selectivity for OX(2) to be useful for determination of receptor subtype expression. Comparison of the potencies of orexin-A and -B with respect to a number of published responses in OX(1)-expressing CHO cells, demonstrates that these show great variation: i.e., orexin-A is 1.6-18-fold more potent than orexin-B, depending on the response assessed. These data together suggest that orexin receptor ligands show signal trafficking, which makes agonist-based pharmacology unreliable. PMID:21362456

1. To evaluate the implication of taurine in the physiology of supraoptic neurones, we (i) investigated the agonist properties of taurine on glycine and GABAA receptors of supraoptic magnocellular neurones acutely dissociated from adult rats, using whole-cell voltage clamp, (ii) studied the effects of taurine and strychnine in vivo by extracellular recordings of supraoptic vasopressin neurones in anaesthetized rats, and (iii) measured the osmolarity-dependent release of endogenous taurine from isolated supraoptic nuclei by HPLC. 2. GABA, glycine and taurine evoked rapidly activating currents that all reversed close to the equilibrium potential for Cl-, indicating activation of Cl(-)-selective channels. Glycine-activated currents were reversibly blocked by strychnine (IC50 of 35 nM with 100 microM glycine), but were unaffected by the GABAA antagonist gabazine (1-3 microM). GABA-activated currents were reversibly antagonized by 3 microM gabazine, but not by strychnine (up to 1 microM). 3. Responses to 1 mM taurine were blocked by strychnine but not by gabazine and showed no additivity with glycine-induced currents, indicating selective activation of glycine receptors. Responses to 10 mM taurine were partially antagonized by gabazine, the residual current being blocked by strychnine. Thus, taurine is also a weak agonist of GABAA receptors. 4. In the presence of gabazine, taurine activated glycine receptors with an EC50 of 406 microM. Taurine activated at most 70% of maximal glycine currents, suggesting that it is a partial agonist of glycine receptors. 5. In vivo, locally applied strychnine (300 nM) increased and taurine (1 mM) decreased the basal electrical activity of vasopressin neurones in normally hydrated rats. The effect of strychnine was markedly more pronounced in water-loaded rats. 6. Taurine, which is concentrated in supraoptic glial cells, could be released from isolated supraoptic nuclei upon hyposmotic stimulation. Decreases in osmolarity of 15 and 30

Microvascular leakage hypersensitivity is a main component of neurogenic inflammation and of tachykinin effects. The aim of this study was to examine the ability of neurokinin B and of the tachykinin NK(3) receptoragonists, [MePhe(7)]neurokinin B or senktide, to potentiate when given by aerosol the microvascular leakage induced by histamine in guinea-pig airways and to compare their effects to those of tachykinin NK(1) (substance P, [Sar(9),Met(O(2))(11)]substance P) or tachykinin NK(2) (neurokinin A, [betaAla(8)]neurokinin A (4-10)) receptoragonists. Guinea-pigs were pretreated successively for 10 min with aerolized salbutamol and phosphoramidon; 15 min later, they were exposed for 30 min to an aerosolized solution of tachykinin receptoragonists; 24 h later, the animals were anaesthetized and vascular permeability was quantified by extravasation of Evans blue dye. Neurokinin B, [MePhe(7)]neurokinin B and senktide (3 x 10(-6)-3 x 10(-5)M) induced a potentiation of the effects of histamine on the vascular permeability in the trachea and main bronchi. Compared to other tachykinin NK(1) and NK(2) receptoragonists, the order of potency was: senktide>neurokinin B=[Sar(9),Met(O(2))(11)]substance P=[betaAla(8)]neurokinin A (4-10)=[MePhe(7)]neurokinin B>neurokinin A>substance P. The potentiation by [MePhe(7)]neurokinin B of histamine-induced microvascular leakage was abolished by the tachykinin NK(1) receptor antagonist SR140333 ([(S)1-(2-[3-(3,4-dichlorophenyl)-1-(3-iso-propoxyphenylacetyl)piperidin-3-yl]etyl)-4-phenyl-1-azoniabicyclo[2.2.2]octane, chloride]) or the tachykinin NK(3) receptor antagonists SR 142801 ([(R)-(N)-(1-(3-(l-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl) propyl)-4-phenylpiperidin-4-yl)-N-methylacetamide]) and SB 223412 ([(S)-(-)-N-(alpha-ethylbenzyl)-3-hydroxy-2-phenylquinoline-4-carboxamide]). In conclusion, these results suggest that tachykinin NK(3) receptors might be involved in the potentiation of histamine-induced increase in microvascular

The P2Y14R is a G(i/o)-coupled receptor of the P2Y family of purinergic receptors that is activated by extracellular UDP and UDP-glucose (UDPG). In an earlier report we described a P2Y14R fluorescent probe, MRS4174, based on the potent and selective antagonist PPTN, a naphthoic acid derivative. Here, we report the design, preparation, and activity of an agonist-based fluorescent probe MRS4183 (11) and a shorter P2Y14R agonist congener, which contain a UDP-glucuronic acid pharmacophore and BODIPY fluorophores conjugated through diaminoalkyl linkers. The design relied on both docking in a P2Y14R homology model and established structure activity relationship (SAR) of nucleotide analogs. 11 retained P2Y14R potency with EC50 value of 0.96 nM (inhibition of adenylyl cyclase), compared to parent UDPG (EC50 47 nM) and served as a tracer for microscopy and flow cytometry, displaying minimal nonspecific binding. Binding saturation analysis gave an apparent binding constant for 11 in whole cells of 21.4±1.1 nM, with a t1/2 of association at 50 nM 11 of 23.9 min. Known P2Y14R agonists and PPTN inhibited cell binding of 11 with the expected rank order of potency. The success in the identification of a new P2Y14R fluorescent agonist with low nonspecific binding illustrates the advantages of rational design based on recently determined GPCR X-ray structures. Such conjugates will be useful tools in expanding the SAR of this receptor, which still lacks chemical diversity in its collective ligands. PMID:26303895

Among the opioid receptors, the κ-opioid receptor (κOR) has been gaining considerable attention as a potential therapeutic target for the treatment of complex CNS disorders including depression, visceral pain, and cocaine addiction. With an interest in discovering novel ligands targeting κOR, we searched natural products for unusual scaffolds and identified collybolide (Colly), a nonnitrogenous sesquiterpene from the mushroom Collybia maculata. This compound has a furyl-δ-lactone core similar to that of Salvinorin A (Sal A), another natural product from the plant Salvia divinorum Characterization of the molecular pharmacological properties reveals that Colly, like Sal A, is a highly potent and selective κOR agonist. However, the two compounds differ in certain signaling and behavioral properties. Colly exhibits 10- to 50-fold higher potency in activating the mitogen-activated protein kinase pathway compared with Sal A. Taken with the fact that the two compounds are equipotent for inhibiting adenylyl cyclase activity, these results suggest that Colly behaves as a biased agonist of κOR. Behavioral studies also support the biased agonistic activity of Colly in that it exhibits ∼10-fold higher potency in blocking non-histamine-mediated itch compared with Sal A, and this difference is not seen in pain attenuation by these two compounds. These results represent a rare example of functional selectivity by two natural products that act on the same receptor. The biased agonistic activity, along with an easily modifiable structure compared with Sal A, makes Colly an ideal candidate for the development of novel therapeutics targeting κOR with reduced side effects. PMID:27162327

The constitutive androstane receptor (CAR, NR1I3) is very important for drug development and for understanding pharmacokinetic drug-drug interactions. We screened by mammalian one hybrid assay among natural compounds to discover novel ligands of human constitutive androstane receptor (hCAR). hCAR transcriptional activity was measured by luciferase assay and mRNA levels of CYP2B6 and CYP3A4 in HepTR-hCAR cells and human primary hepatocytes were measured by real-time RT-PCR. Nigramide J (NJ) whose efficacy is comparable to those of hitherto known inverse agonists such as clotrimazole, PK11195, and ethinylestradiol. NJ is a naturally occurring cyclohexane-type amide alkaloid that was isolated from the roots of Piper nigrum. The suppressive effect of NJ on the CAR-dependent transcriptional activity was found to be species specific, in the descending order of hCAR, rat CAR, and mouse CAR. The unliganded hCAR-dependent transactivation of reporter and endogenous genes was suppressed by NJ at concentrations higher than 5 μmol/L. The ligand-binding cavity of hCAR was shared by NJ and CITCO, because they were competitive in the binding to hCAR. NJ interfered with the interaction of hCAR with coactivator SRC-1, but not with its interaction with the corepressor NCoR1. Furthermore, NJ is agonist of human pregnane X receptor (hPXR). NJ is a dual ligand of hCAR and hPXR, being an agonist of hPXR and an inverse agonist of hCAR. PMID:25505573

BACKGROUND AND PURPOSE The transient receptor potential vanilloid 1 (TRPV1) plays a role in the activation of sensory neurons by various painful stimuli and is a therapeutic target. However, functional TRPV1 that affect microvascular diameter are also expressed in peripheral arteries and we attempted to characterize this receptor. EXPERIMENTAL APPROACH Sensory TRPV1 activation was measured in rats by use of an eye wiping assay. Arteriolar TRPV1-mediated smooth muscle specific responses (arteriolar diameter, changes in intracellular Ca2+) were determined in isolated, pressurized skeletal muscle arterioles obtained from the rat and wild-type or TRPV1−/− mice and in canine isolated smooth muscle cells. The vascular pharmacology of the TRPV1 agonists (potency, efficacy, kinetics of action and receptor desensitization) was determined in rat isolated skeletal muscle arteries. KEY RESULTS Capsaicin evoked a constrictor response in isolated arteries similar to that mediated by noradrenaline, this was absent in arteries from TRPV1 knockout mice and competitively inhibited by TRPV1 antagonist AMG9810. Capsaicin increased intracellular Ca2+ in the arteriolar wall and in isolated smooth muscle cells. The TRPV1 agonists evoked similar vascular constrictions (MSK-195 and JYL-79) or were without effect (resiniferatoxin and JYL-273), although all increased the number of responses (sensory activation) in the eye wiping assay. Maximal doses of all agonists induced complete desensitization (tachyphylaxis) of arteriolar TRPV1 (with the exception of capsaicin). Responses to the partial agonist JYL-1511 suggested 10% TRPV1 activation is sufficient to evoke vascular tachyphylaxis without sensory activation. CONCLUSIONS AND IMPLICATIONS Arteriolar TRPV1 have different pharmacological properties from those located on sensory neurons in the rat. PMID:21883148

Chemical feature based pharmacophore models were generated for Toll-like receptors 7 (TLR7) agonists using HypoGen algorithm, which is implemented in the Discovery Studio software. Several methods tools used in validation of pharmacophore model were presented. The first hypothesis Hypo1 was considered to be the best pharmacophore model, which consists of four features: one hydrogen bond acceptor, one hydrogen bond donor, and two hydrophobic features. In addition, homology modeling and molecular docking studies were employed to probe the intermolecular interactions between TLR7 and its agonists. The results further confirmed the reliability of the pharmacophore model. The obtained pharmacophore model (Hypo1) was then employed as a query to screen the Traditional Chinese Medicine Database (TCMD) for other potential lead compounds. One hit was identified as a potent TLR7 agonist, which has antiviral activity against hepatitis virus in vitro. Therefore, our current work provides confidence for the utility of the selected chemical feature based pharmacophore model to design novel TLR7 agonists with desired biological activity. PMID:23526932

Allogeneic transplantation can cure many disorders, including sickle cell disease, chronic granulomatous disease (CGD), severe combined immunodeficiency (SCID) and many types of cancers. However, there are several associated risks that can result in severe immunological reactions and, in some cases, death. Much of this morbidity is related to graft versus host disease (GVHD) [1]. GVHD is an immune mediated reaction in which donor T cells recognize the host as antigenically foreign, causing donor T cells to expand and attack host tissues. The current method of treating recent transplant patients with immunosuppressants to prevent this reaction has met with only partial success, emphasizing a need for new methods of GVHD treatment and prevention. Recently, a novel strategy has emerged targeting adenosine A2A receptors (A2AR) through the use of adenosine agonists. These agonists have been shown in vitro to increase the TGFβ-induced generation of FoxP3(+) regulatory T cells (Tregs) and in vivo to improve weight gain and mortality as well as inhibit the release of pro-inflammatory cytokines in GVHD murine models [2,3]. Positive results involving A2AR agonists in vitro and in vivo are promising, suggesting that A2AR agonists should be a part of the management of clinical GvHD. PMID:25709759

Liver X receptor α (LXRα) plays an important role in the cholesterol metabolism process, and LXRα activation can reduce atherosclerosis. In the present study, using an LXRα-GAL4 luciferase reporter screening, we discovered IMB-170, a structural analog of quinazolinone, which showed potent LXRα agonistic activity. IMB-170 significantly activated LXRα, with an EC50 value of 0.27μM. Interestingly, IMB-170 not only increased the expression of ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1), which are related to the reverse cholesterol transport (RCT) process, but also influenced the expression levels of other genes involved in the cholesterol metabolism pathway in many cell lines. Moreover, IMB-170 significantly reduced cellular lipid accumulation and increased cholesterol efflux from RAW264.7 and THP-1 macrophages. Interestingly, compared with TO901317, IMB-170 only slightly increased protein expression levels of lipogenesis-related genes in HepG2 cells, indicating that IMB-170 may have a lower lipogenesis side effect in vivo. These results suggest that IMB-170 showed the selective agonistic activity for LXRα. Moreover, compared with full LXR-agonists, IMB-170 possesses a differential ability to recruit coregulators. This suggests that IMB-170 has distinct interactions with the active sites in the LXRα ligand-binding domain. In summary, IMB-170 is a novel partial LXRα agonist without the classical lipogenesis side effects, which could be used as a potential anti-atherosclerotic leading compound in the future. PMID:25450668

The vanilloid receptor TRPV1 is a homotetrameric, non-selective cation channel abundantly expressed in the nociceptors (c-fibers). TRPV1 is considered as a highly validated pain target because, i) its agonists such as capsaicin cause desensitization of TRPV1 channels that relieves pain behaviors in preclinical species, and ii) its antagonists relieve pain behaviors in rodent models of inflammation, osteoarthritis, and cancer. Hence, both agonists and antagonists of TRPV1 are being evaluated as potential analgesics in clinical trials. Clinical trial results of TRPV1 agonists such as resiniferatoxin in interstitial cystitis, NGX 4010 in post-herpetic neuralgia, and 4975 (Adlea) in osteoarthritis, bunionectomy, and Morton's neuroma have been reported. Similarly, clinical trial results of TRPV1 antagonists such as SB-705498 and AMG 517 have also been published recently. Overall, some molecules (e.g., capsaicin) demonstrated potential analgesia in certain conditions (postsurgical pain, postherpetic neuralgia, pain in diabetic neuropathy, osteoarthritis, bunionectomy, and Morton's neuroma), whereas others fell out of the clinic due to on-target liabilities or failed to demonstrate efficacy. This review summarizes recent advances and setbacks of TRPV1 agonists and antagonists in the clinic and predicts future directions. PMID:19150372

Zebrafish (Danio rerio) embryos were comparably exposed to seven known agonists of retinoid X receptors (RXRs) including two endogenous compounds (9-cis-retinoic acid and docosahexaenoic acid), four man-made selective ligands (LGD1069, SR11237, fluorobexarotene and CD3254), and a biocide (triphenyltin). The dominant phenotypes of malformation were sharp mouths and small caudal fins in 1 mg/L SR11237-treated group after 5 days exposure. 9-cis-retinoic acid and LGD1069 induced multiple malformations including small eyes, bent notochords, reduced brain, enlarged proctodaems, absence of fins, short tails and edema after 5 days exposure. Fluorobexarotene and CD3254 induced similar phenotypes of malformations after 5 days exposure at low concentration (20 μg/L) to those after the 1st d exposure at high concentrations (50 and 100 μg/L). Triphenlytin induced multiple malformations including deformed eyes, bent notochords, bent tails, and edema in hearts after 5 days exposure at concentrations of 1-10 μg Sn/L. In contrast, no discernible malformations were observed in triphenlytin-treated groups after each separate day exposure. These agonists not only showed different ability of teratogenicity but also induced different phenotypes of malformation in zebrafish embryos. In addition, the sensitive stages of zebrafish embryos were different in response to these agonists. Therefore, our results suggest that the agonists of RXRs had divergent teratogenicity in zebrafish embryos. PMID:22526925

Type 2 diabetes (T2D) occurs when there is insufficient insulin release to control blood glucose, due to insulin resistance and impaired β-cell function. The GPR39 receptor is expressed in metabolic tissues including pancreatic β-cells and has been proposed as a T2D target. Specifically, GPR39 agonists might improve β-cell function leading to more adequate and sustained insulin release and glucose control. The present study aimed to test the hypothesis that GPR39 agonism would improve glucose stimulated insulin secretion in vivo. A high throughput screen, followed by a medicinal chemistry program, identified three novel potent Zn2+ modulated GPR39 agonists. These agonists were evaluated in acute rodent glucose tolerance tests. The results showed a lack of glucose lowering and insulinotropic effects not only in lean mice, but also in diet-induced obese (DIO) mice and Zucker fatty rats. It is concluded that Zn2+ modulated GPR39 agonists do not acutely stimulate insulin release in rodents. PMID:26720709

Drugs containing adrenocorticotropic hormone have been used as therapy for patients with nephrotic syndrome. We have previously shown that adrenocorticotropic hormone and a selective agonist for the melanocortin 1 receptor (MC1R) exert beneficial actions in experimental membranous nephropathy with reduced proteinuria, reduced oxidative stress, and improved glomerular morphology and function. Our hypothesis is that MC1R activation in podocytes elicits beneficial effects by promoting stress fibers and maintaining podocyte viability. To test the hypothesis, we cultured podocytes and used highly specific agonists for MC1R. Podocytes were subjected to the nephrotic-inducing agent puromycin aminonucleoside, and downstream effects of MC1R activation on podocyte survival, antioxidant defense, and cytoskeleton dynamics were studied. To increase the response and enhance intracellular signals, podocytes were transduced to overexpress MC1R. We showed that puromycin promotes MC1R expression in podocytes and that activation of MC1R promotes an increase of catalase activity and reduces oxidative stress, which results in the dephosphorylation of p190RhoGAP and formation of stress fibers through RhoA. In addition, MC1R agonists protect against apoptosis. Together, these mechanisms protect the podocyte against puromycin. Our findings strongly support the hypothesis that selective MC1R-activating agonists protect podocytes and may therefore be useful to treat patients with nephrotic syndromes commonly considered as podocytopathies. PMID:26887829

Objective Type 2 diabetes and obesity are emerging pandemics in the 21st century creating worldwide urgency for the development of novel and safe therapies. We investigated trace amine-associated receptor 1 (TAAR1) as a novel target contributing to the control of glucose homeostasis and body weight. Methods We investigated the peripheral human tissue distribution of TAAR1 by immunohistochemistry and tested the effect of a small molecule TAAR1 agonist on insulin secretion in vitro using INS1E cells and human islets and on glucose tolerance in C57Bl6, and db/db mice. Body weight effects were investigated in obese DIO mice. Results TAAR1 activation by a selective small molecule agonist increased glucose-dependent insulin secretion in INS1E cells and human islets and elevated plasma PYY and GLP-1 levels in mice. In diabetic db/db mice, the TAAR1 agonist normalized glucose excursion during an oral glucose tolerance test. Sub-chronic treatment of diet-induced obese (DIO) mice with the TAAR1 agonist resulted in reduced food intake and body weight. Furthermore insulin sensitivity was improved and plasma triglyceride levels and liver triglyceride content were lower than in controls. Conclusions We have identified TAAR1 as a novel integrator of metabolic control, which acts on gastrointestinal and pancreatic islet hormone secretion. Thus TAAR1 qualifies as a novel and promising target for the treatment of type 2 diabetes and obesity. PMID:26844206

In a previous communication we reported on the discovery of alkylamino pyridine derivatives (e.g. 1) as a new class of potent, selective and efficacious S1P1 receptor (S1PR1) agonists. However, more detailed profiling revealed that this compound class is phototoxic in vitro. Here we describe a new class of potent S1PR1 agonists wherein the exocyclic nitrogen was moved away from the pyridine ring (e.g. 11c). Further structural modifications led to the identification of novel alkylaminomethyl substituted phenyl and thienyl derivatives as potent S1PR1 agonists. These new alkylaminomethyl aryl compounds showed no phototoxic potential. Based on their in vivo efficacy and ability to penetrate the brain, the 5-alkyl-aminomethyl thiophenes appeared to be the most interesting class. Potent and selective S1PR1 agonist 20e, for instance, maximally reduced the blood lymphocyte count (LC) for 24 h after oral administration of 10 mg/kg to rat and its brain concentrations reached >500 ng/g over 24 h. PMID:27061986

Endocrine disrupting chemicals with estrogenic activity (EA) have been associated with various adverse health effects. US agencies (ICCVAM/NICEATM) tasked to assess in vitro transcription activation assays to detect estrogenic receptor (ER) agonists for EA have recently validated a BG1Luc assay in manual format, but prefer robotic formats. We have developed a robotic BG1Luc EA assay to detect EA that demonstrated 100% concordance with ICCVAM meta-analyses and ICCVAM BG1Luc results in manual format for 27 ICCVAM test substances, i.e. no false negatives or false positives. This robotic assay also consistently assessed other, more problematic ICCVAM test substances such as clomiphene citrate, L-thyroxin, and tamoxifen. Agonist responses using this robotic BG1Luc assay were consistently inhibited by the ER antagonist ICI 182,780, confirming that agonist responses were due to binding to ERs rather than to a non-specific agonist response. This robotic assay also detected EA in complex mixtures of substances such as extracts of personal care products, plastic resins or plastic consumer products. This robotic BG1Luc assay had at least as high accuracy and greater sensitivity and repeatability when compared to its manual version or to the other ICCVAM/OECD validated assays for EA (manual BG1Luc and CERI). PMID:24747293

The kappa opioid receptor (KOR) is widely expressed in the CNS and can serve as a means to modulate pain perception, stress responses, and affective reward states. Therefore, the KOR has become a prominent drug discovery target toward treating pain, depression, and drug addiction. Agonists at KOR can promote G protein coupling and βarrestin2 recruitment as well as multiple downstream signaling pathways, including ERK1/2 MAPK activation. It has been suggested that the physiological effects of KOR activation result from different signaling cascades, with analgesia being G protein-mediated and dysphoria being mediated through βarrestin2 recruitment. Dysphoria associated with KOR activation limits the therapeutic potential in the use of KOR agonists as analgesics; therefore, it may be beneficial to develop KOR agonists that are biased toward G protein coupling and away from βarrestin2 recruitment. Here, we describe two classes of biased KOR agonists that potently activate G protein coupling but weakly recruit βarrestin2. These potent and functionally selective small molecule compounds may prove to be useful tools for refining the therapeutic potential of KOR-directed signaling in vivo. PMID:24187130

The serotonin 5-HT2A receptor is a primary target of psychedelic hallucinogens such as lysergic acid diethylamine, mescaline, and psilocybin, which reproduce some of the core symptoms of schizophrenia. An incompletely resolved paradox is that only some 5-HT2A receptoragonists exhibit hallucinogenic activity, whereas structurally related agonists with comparable affinity and activity lack such a psychoactive activity. Using a strategy combining stable isotope labeling by amino acids in cell culture with enrichment in phosphorylated peptides by means of hydrophilic interaction liquid chromatography followed by immobilized metal affinity chromatography, we compared the phosphoproteome in HEK-293 cells transiently expressing the 5-HT2A receptor and exposed to either vehicle or the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) or the nonhallucinogenic 5-HT2A agonist lisuride. Among the 5995 identified phosphorylated peptides, 16 sites were differentially phosphorylated upon exposure of cells to DOI versus lisuride. These include a serine (Ser280) located in the third intracellular loop of the 5-HT2A receptor, a region important for its desensitization. The specific phosphorylation of Ser280 by hallucinogens was further validated by quantitative mass spectrometry analysis of immunopurified receptor digests and by Western blotting using a phosphosite specific antibody. The administration of DOI, but not of lisuride, to mice, enhanced the phosphorylation of 5-HT2A receptors at Ser280 in the prefrontal cortex. Moreover, hallucinogens induced a less pronounced desensitization of receptor-operated signaling in HEK-293 cells and neurons than did nonhallucinogenic agonists. The mutation of Ser280 to aspartic acid (to mimic phosphorylation) reduced receptor desensitization by nonhallucinogenic agonists, whereas its mutation to alanine increased the ability of hallucinogens to desensitize the receptor. This study reveals a biased phosphorylation of

The serotonin 5-HT(2A) receptor is a primary target of psychedelic hallucinogens such as lysergic acid diethylamine, mescaline, and psilocybin, which reproduce some of the core symptoms of schizophrenia. An incompletely resolved paradox is that only some 5-HT(2A) receptoragonists exhibit hallucinogenic activity, whereas structurally related agonists with comparable affinity and activity lack such a psychoactive activity. Using a strategy combining stable isotope labeling by amino acids in cell culture with enrichment in phosphorylated peptides by means of hydrophilic interaction liquid chromatography followed by immobilized metal affinity chromatography, we compared the phosphoproteome in HEK-293 cells transiently expressing the 5-HT(2A) receptor and exposed to either vehicle or the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) or the nonhallucinogenic 5-HT(2A) agonist lisuride. Among the 5995 identified phosphorylated peptides, 16 sites were differentially phosphorylated upon exposure of cells to DOI versus lisuride. These include a serine (Ser(280)) located in the third intracellular loop of the 5-HT(2A) receptor, a region important for its desensitization. The specific phosphorylation of Ser(280) by hallucinogens was further validated by quantitative mass spectrometry analysis of immunopurified receptor digests and by Western blotting using a phosphosite specific antibody. The administration of DOI, but not of lisuride, to mice, enhanced the phosphorylation of 5-HT(2A) receptors at Ser(280) in the prefrontal cortex. Moreover, hallucinogens induced a less pronounced desensitization of receptor-operated signaling in HEK-293 cells and neurons than did nonhallucinogenic agonists. The mutation of Ser(280) to aspartic acid (to mimic phosphorylation) reduced receptor desensitization by nonhallucinogenic agonists, whereas its mutation to alanine increased the ability of hallucinogens to desensitize the receptor. This study reveals a biased

Although Δ(9)-tetrahydrocannabinol (THC) and other mixed CB(1)/CB(2) receptoragonists are well established to elicit antinociceptive effects, their psychomimetic actions and potential for abuse have dampened enthusiasm for their therapeutic development. Conversely, CB(2) receptor-selective agonists have been shown to reduce pain and inflammation, without eliciting apparent cannabinoid behavioral effects. In the present study, we developed a novel ethyl sulfonamide THC analog, O-3223, and compared its pharmacological effects to those of the potent, mixed CB(1)/CB(2) receptoragonist, CP55,940, in a battery of preclinical pain models. Competitive cannabinoid receptor binding experiments revealed that O-3223 was approximately 80-fold more selective for CB(2) than CB(1) receptors. Additionally, O-3223 behaved as a full CB(2) receptoragonist in [(35)S]GTPγS binding. O-3223 reduced nociceptive behavior in both phases of the formalin test, reduced thermal hyperalgesia in the chronic constriction injury of the sciatic nerve (CCI) model, and reduced edema and thermal hyperalgesia elicited by intraplantar injection of LPS. These effects were blocked by pretreatment with the CB(2) receptor-selective antagonist SR144528, but not by the CB(1) receptor antagonist, rimonabant. Unlike CP55,940, O-3223 did not elicit acute antinociceptive effects in the hot-plate test, hypothermia, or motor disturbances, as assessed in the rotarod test. These data indicate that the CB(2) receptor-selective agonist, O-3223, reduces inflammatory and neuropathic nociception, without affecting basal nociception or eliciting overt behavioral effects. Moreover, this compound can serve as a template to develop new CB(2) receptoragonists with increased receptor selectivity and increased potency in treating inflammatory and neuropathic pain. PMID:20849866

Darmstoff describes a family of gut smooth muscle-stimulating acetal phosphatidic acids initially isolated and characterized from the bath fluid of stimulated gut over 50 years ago. Despite similar structural and biological profiles, Darmstoff analogs have not previously been examined as potential LPA mimetics. Here, we report a facile method for the synthesis of potassium salts of Darmstoff analogs. To understand the effect of stereochemistry on lysophosphatidic acid mimetic activity, synthesis of optically pure stereoisomers of selected Darmstoff analogs was achieved starting with chiral methyl glycerates. Each Darmstoff analog was evaluated for subtype-specific LPA receptoragonist/antagonist activity, PPARgamma activation, and autotaxin inhibition. From this study we identified compound 12 as a pan-antagonist and several pan-agonists for the LPA(1-3) receptors. Introduction of an aromatic ring in the lipid chain such as analog 22 produced a subtype-specific LPA(3) agonist with an EC(50) of 692 nM. Interestingly, regardless of their LPA(1/2/3) ligand properties all of the Darmstoff analogs tested activated PPARgamma. However, these compounds are weak inhibitors of autotaxin. The results indicate that Darmstoff analogs constitute a novel class of lysophosphatidic acid mimetics. PMID:16290140

BW723C86, a serotonin receptor 2B agonist, has been investigated as a potential therapeutic for various conditions such as anxiety, hyperphagia and hypertension. However, the functional role of BW723C86 against melanogenesis remains unclear. In this study, we investigate the effect of serotonin receptor 2B (5-HTR2B) agonist on melanogenesis and elucidate the mechanism involved. BW723C86 reduced melanin synthesis and intracellular tyrosinase activity in melan-A cells and normal human melanocytes. The expression of melanogenesis-related proteins (tyrosinase, TRP-1 and TRP-2) and microphthalmia-associated transcription factor (MITF) in melan-A cells decreased after BW723C86 treatment. The promoter activity of MITF was also reduced by BW723C86 treatment. The reduced level of MITF was associated with inhibition of protein kinase A (PKA) and cAMP response element-binding protein (CREB) activation by BW723C86 treatment. These results suggest that the serotonin agonist BW723C86 could be a potential therapeutic agent for skin hyperpigmentation disorders. PMID:27077852

Neurotensin (NT) is an endogenous tridecapeptide found in the central nervous system (CNS) and in peripheral tissues. Neurotensin exerts a wide range of physiological effects and it has been found to play a critical role in a number of human diseases, such as schizophrenia, Parkinson’s disease and drug addiction. The discovery of small-molecule non-peptide neurotensin receptor (NTSR) modulators would represent an important breakthrough as such compounds could be used as pharmacological tools, to further decipher the cellular functions of neurotensin, and potentially as therapeutic agents to treat human disease. Herein, we report the identification of non-peptide low-micromolar neurotensin receptor 1 (NTSR1) full agonists, discovered through structural optimization of the known NTSR1 partial agonist 1. In vitro cellular screenings, based on an intracellular Ca2+ mobilization assay, revealed our best hit molecule 8 (SR-12062) to have an EC50 of 2 μM at NTSR1 with full agonist behaviour (Emax = 100%), showing a higher efficacy and ~ 90-fold potency improvement compared to parent compound 1 (EC50 = 178 μM; Emax = 17%). PMID:24997685

BW723C86, a serotonin receptor 2B agonist, has been investigated as a potential therapeutic for various conditions such as anxiety, hyperphagia and hypertension. However, the functional role of BW723C86 against melanogenesis remains unclear. In this study, we investigate the effect of serotonin receptor 2B (5-HTR2B) agonist on melanogenesis and elucidate the mechanism involved. BW723C86 reduced melanin synthesis and intracellular tyrosinase activity in melan-A cells and normal human melanocytes. The expression of melanogenesis-related proteins (tyrosinase, TRP-1 and TRP-2) and microphthalmia-associated transcription factor (MITF) in melan-A cells decreased after BW723C86 treatment. The promoter activity of MITF was also reduced by BW723C86 treatment. The reduced level of MITF was associated with inhibition of protein kinase A (PKA) and cAMP response element-binding protein (CREB) activation by BW723C86 treatment. These results suggest that the serotonin agonist BW723C86 could be a potential therapeutic agent for skin hyperpigmentation disorders. PMID:27077852

We hypothesized that people with cystic fibrosis (CF) who express CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations associated with residual function may benefit from G-protein coupled receptor (GPCR)-targeting drugs that can activate and enhance CFTR function.We used intestinal organoids to screen a GPCR-modulating compound library and identified β2-adrenergic receptoragonists as the most potent inducers of CFTR function.β2-Agonist-induced organoid swelling correlated with the CFTR genotype, and could be induced in homozygous CFTR-F508del organoids and highly differentiated primary CF airway epithelial cells after rescue of CFTR trafficking by small molecules. The in vivo response to treatment with an oral or inhaled β2-agonist (salbutamol) in CF patients with residual CFTR function was evaluated in a pilot study. 10 subjects with a R117H or A455E mutation were included and showed changes in the nasal potential difference measurement after treatment with oral salbutamol, including a significant improvement of the baseline potential difference of the nasal mucosa (+6.35 mV, p<0.05), suggesting that this treatment might be effective in vivo Furthermore, plasma that was collected after oral salbutamol treatment induced CFTR activation when administered ex vivo to organoids.This proof-of-concept study suggests that organoids can be used to identify drugs that activate CFTR function in vivo and to select route of administration. PMID:27471203

An epidemic of metabolic diseases including type 2 diabetes and obesity is undermining the health of people living in industrialized societies. There is an urgent need to develop innovative therapeutics. The peroxisome proliferator-activated receptor gamma (PPARgamma) is one of the ligand-activated transcription factors in the nuclear hormone receptor superfamily and a pivotal regulator of glucose and lipid homeostasis. The discovery of PPARgamma as a target of multimodal insulin sensitizers, represented by thiazolidinediones (TZDs), has attracted remarkable scientific interest and had a great impact on the pharmaceutical industry. With the clinical success of the PPARgamma agonists, pioglitazone (Actos) and rosiglitazone (Avandia), development of novel and potent insulin-sensitizing agents with diverse clinical profiles has been accelerated. Currently, a number of PPARgamma agonists from different chemical classes and with varying pharmacological profiles are being developed. Despite quite a few obstacles to the development of PPAR-related drugs, PPARgamma-targeted agents still hold promise. There are new concepts and encouraging evidence emerging that suggest this class can yield improved anti-diabetic agents. This review covers the discovery of TZDs, provides an overview of PPARgamma including the significance of PPARgamma as a drug target, describes the current status of a wide variety of novel PPARgamma ligands including PPAR dual and pan agonists and selective PPARgamma modulators (SPPARgammaMs), and highlights new approaches for identifying agents targeting PPARgamma in the treatment of type 2 diabetes. PMID:19075761

In preceding communications we summarized our medicinal chemistry efforts leading to the identification of potent, selective, and orally active S1P1 agonists such as the thiophene derivative 1. As a continuation of these efforts, we replaced the thiophene in 1 by a 2-, 3-, or 4-pyridine and obtained less lipophilic, potent, and selective S1P1 agonists (e.g., 2) efficiently reducing blood lymphocyte count in the rat. Structural features influencing the compounds' receptor affinity profile and pharmacokinetics are discussed. In addition, the ability to penetrate brain tissue has been studied for several compounds. As a typical example for these pyridine based S1P1 agonists, compound 53 showed EC50 values of 0.6 and 352 nM for the S1P1 and S1P3 receptor, respectively, displayed favorable PK properties, and penetrated well into brain tissue. In the rat, compound 53 maximally reduced the blood lymphocyte count for at least 24 h after oral dosing of 3 mg/kg. PMID:24367923

Monoclonal antibody M-35, which immunoprecipitates native calf brain acetylcholine muscarinic receptor, mimics agonist stimulation of the intact guinea pig myometrium: the antibody, just like carbamoylcholine hydrochloride, causes a rise in intracellular cyclic GMP content, an inhibition of cyclic AMP accumulation due to prostacyclin, and induces uterine contractions. Another antibody, M-23, which reacts with the denatured muscarinic receptor, is devoid of agonist-like activity at the cyclic nucleotide level but is still able to induce contractions of both rat and guinea pig myometrium. The cyclic nucleotide changes caused by both carbamoylcholine and antibody M-35 are inhibited by atropine; this antagonist, which blocks carbamoylcholine-mediated contractions, fails however, to prevent contractions induced by antibodies M-35 and M-23. These results suggest that the information necessary to transmit muscarinic signals is entirely contained in the receptor and that ligands only act to trigger the biological response. The data also imply that the muscarinic receptors of the myometrium are coupled to multiple effector systems. PMID:6087318

Anti-parkinsonian agents, pramipexole (PPX) and ropinirole (ROP), have been reported to possess neuroprotective properties, both in vitro and in vivo. The mechanisms underlying neuroprotection afforded by the D3-preferring receptoragonists remain poorly understood. The present study demonstrates that incubation of primary mesencephalic cultures with PPX and ROP or the conditioned medium from PPX- or ROP-treated primary cultures induced a marked increase in the number of dopamine (DA) neurons in the cultures. Similar effects can be observed after incubating with the conditioned medium derived from PPX- and ROP-treated substantia nigra astroglia. Meanwhile, PPX and ROP can protect the primary cells from insult of 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP). Furthermore, the neurotrophic effects of PPX and ROP on mesencephalic dopamine neurons could be significantly blocked by D3 receptor antagonist, but not by D2 receptor antagonist. Moreover, we found that the levels of glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) in the conditioned medium of mesencephalic cultures treated with PPX and ROP were significantly increased. Blocking GDNF and BDNF with the neutralizing antibodies, the neurotrophic effects of PPX and ROP were greatly diminished. These results suggest that D3 dopamine receptor-preferring agonists, PPX and ROP, exert neurotrophic effects on cultured DA neurons by modulating the production of endogenous GDNF and BDNF, which may participate in their neuroprotection. PMID:16307585

The in vitro estrogen receptor (ER) agonist and androgen receptor (AR) antagonist potencies of offshore produced water effluents collected from the Norwegian Sector were determined using recombinant yeast estrogen and androgen screens. Solid phase extraction (SPE) concentrates of the effluents showed E2 agonist activities similar to those previously reported for the United Kingdom (UK) Continental Shelf (<0.1-4 ng E2 L(-1)). No activity was detected in the filtered oil droplets suggesting that produced water ER activity is primarily associated with the dissolved phase. Targeted analysis for methyl- to nonyl-substituted alkylphenol isomers show the occurrence of known ER agonists in the analysed samples. For the first time, AR antagonists were detected in both the dissolved and oil associated phase at concentrations of between 20 and 8000 microg of flutamide equivalents L(-1). The identity of the AR antagonists is unknown, however this represents a significant input into the marine environment of unknown compounds that exert a known biological effect. It is recommended that further analysis using techniques such as bioassay-directed analysis is performed to identify the compounds/groups of compounds that are responsible in order to improve the assessment of the risk posed by produced water discharges to the marine environment. PMID:17258235

The recruitment of T lymphocytes during diseases such as rheumatoid arthritis is regulated by stimulation of the chemokine receptors expressed by these cells. This study was designed to assess the potential of a CXCR3-specific small-molecule agonist to inhibit the migration of activated human T cells toward multiple chemokines. Further experiments defined the molecular mechanism for this anti-inflammatory activity. Analysis in vitro demonstrated agonist induced internalization of both CXCR3 and other chemokine receptors coexpressed by CXCR3(+) T cells. Unlike chemokine receptor-specific antagonists, the CXCR3 agonist inhibited migration of activated T cells toward the chemokine mixture in synovial fluid from patients with active rheumatoid arthritis. A humanized mouse air-pouch model showed that intravenous treatment with the CXCR3 agonist prevented inflammatory migration of activated human T cells toward this synovial fluid. A potential mechanism for this action was defined by demonstration that the CXCR3 agonist induces receptor cross-phosphorylation within CXCR3-CCR5 heterodimers on the surface of activated T cells. This study shows that generalized chemokine receptor desensitization can be induced by specific stimulation of a single chemokine receptor on the surface of activated human T cells. A humanized mouse model was used to demonstrate that this receptor desensitization inhibits the inflammatory response that is normally produced by the chemokines present in synovial fluid from patients with active rheumatoid arthritis. PMID:22392992

Background and purpose: There is growing interest in using cannabinoid type 2 (CB2) receptoragonists for the treatment of neuropathic pain. In this report, we describe the pharmacological characteristics of MDA7 (1-[(3-benzyl-3-methyl-2,3-dihydro-1-benzofuran-6-yl)carbonyl]piperidine), a novel CB2 receptoragonist. Experimental approach: We characterized the pharmacological profile of MDA7 by using radioligand-binding assays and in vitro functional assays at human cannabinoid type 1 (CB1) and CB2 receptors. In vitro functional assays were performed at rat CB1 and CB2 receptors. The effects of MDA7 in reversing neuropathic pain were assessed in spinal nerve ligation and paclitaxel-induced neuropathy models in rats. Key results: MDA7 exhibited selectivity and agonist affinity at human and rat CB2 receptors. MDA7 treatment attenuated tactile allodynia produced by spinal nerve ligation or by paclitaxel in a dose-related manner. These effects were selectively antagonized by a CB2 receptor antagonist but not by CB1 or opioid receptor antagonists. MDA7 did not affect rat locomotor activity. Conclusion and implications: MDA7, a novel selective CB2 agonist, was effective in suppressing neuropathic nociception in two rat models without affecting locomotor behaviour. These results confirm the potential for CB2 agonists in the treatment of neuropathic pain. PMID:18846037

The pleiotropic effects of retinoic acid on cell differentiation and proliferation are mediated by two subfamilies of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Recently the synthetic retinoid Ro 41-5253 was identified as a selective RAR alpha antagonist. As demonstrated by gel retardation assays, Ro 41-5253 and two related new RAR alpha antagonists do not influence RAR alpha/RXR alpha heterodimerization and DNA binding. In a limited trypsin digestion assay, complexation of RAR alpha with retinoic acid or several other agonistic retinoids altered the degradation of the receptor such that a 30-kDa proteolytic fragment became resistant to proteolysis. This suggests a ligand-induced conformational change, which may be necessary for the interaction of the DNA-bound RAR alpha/RXR alpha heterodimer with other transcription factors. Our results demonstrate that antagonists compete with agonists for binding to RAR alpha and may induce a different structural alteration, suggested by the tryptic resistance of a shorter 25-kDa protein fragment in the digestion assay. This RAR alpha conformation seems to allow RAR alpha/RXR alpha binding to DNA but not the subsequent transactivation of target genes. Protease mapping with C-terminally truncated receptors revealed that the proposed conformational changes mainly occur in the DE regions of RAR alpha. Complexation of RAR beta, RAR gamma, and RXR alpha, as well as the vitamin D3 receptor, with their natural ligands resulted in a similar resistance of fragments to proteolytic digestion. This could mean that ligand-induced conformational changes are a general feature in the hormonal activation of vitamin D3 and retinoid receptors. Images PMID:8264595

Endocannabinoids can affect multiple cellular targets, such as cannabinoid (CB) receptors, transient receptor potential cation channel, subfamily V, member 1 (TRPV1) and peroxisome proliferator-activated receptor γ (PPARγ). The stimuli to induce adipocyte differentiation in hBM-MSCs increase the gene transcription of the CB1 receptor, TRPV1 and PPARγ. In this study, the effects of three endocannabinoids, N-arachidonoyl ethanolamine (AEA), N-arachidonoyl dopamine (NADA) and 2-arachidonoyl glycerol (2-AG), on adipogenesis in hBM-MSCs were evaluated. The adipocyte differentiation was promoted by AEA whereas inhibited by NADA. No change was observed by the treatment of non-cytotoxic concentrations of 2-AG. The difference between AEA and NADA in the regulation of adipogenesis is associated with their effects on PPARγ transactivation. AEA can directly activate PPARγ. The effect of AEA on PPARγ in hBM-MSCs may prevail over that on the CB1 receptor mediated signal transduction, giving rise to the AEA-induced promotion of adipogenesis. In contrast, NADA had no effect on the PPARγ activity in the PPARγ transactivation assay. The inhibitory effect of NADA on adipogenesis in hBM-MSCs was reversed not by capsazepine, a TRPV1 antagonist, but by rimonabant, a CB1 antagonist/inverse agonist. Rimonabant by itself promoted adipogenesis in hBM-MSCs, which may be interpreted as the result of the inverse agonism of the CB1 receptor. This result suggests that the constantly active CB1 receptor may contribute to suppress the adipocyte differentiation of hBM-MSCs. Therefore, the selective CB1 agonists that are unable to affect cellular PPARγ activity inhibit adipogenesis in hBM-MSCs. PMID:25995819

The plant hormone abscisic acid (ABA) plays important roles in regulating plant growth, development, and responses to environmental stresses. Proteins in the PYR/PYL/RCAR family (hereafter referred to as PYLs) are known as ABA receptors. Since most studies thus far have focused on Arabidopsis PYLs, little is known about PYL homologs in crop plants. We report here the characterization of 21 PYL homologs (GmPYLs) in soybean. Twenty three putative GmPYLs can be found from soybean genome sequence and categorized into three subgroups. GmPYLs interact with AtABI1 and two GmPP2Cs in diverse manners. A lot of the subgroup I GmPYLs interact with PP2Cs in an ABA-dependent manner, whereas most of the subgroup II and III GmPYLs bind to PP2Cs in an ABA-independent manner. The subgroup III GmPYL23, which cannot interact with any of the tested PP2Cs, differs from other GmPYLs. The CL2/gate domain is crucial for GmPYLs-PP2Cs interaction, and a mutation in the conserved proline (P109S) abolishes the interaction between GmPYL1 and AtABI1. Furthermore, the ABA dependence of GmPYLs-PP2Cs interactions are partially correlated with two amino acid residues preceding the CL2/gate domain of GmPYLs. We also show that GmPYL1 interacts with AtABI1 in an ABA-dependent manner in plant cells. Three GmPYLs differentially inhibit AtABI1 and GmPP2C1 in an ABA-dependent or -enhanced manner in vitro. In addition, ectopically expressing GmPYL1 partially restores ABA sensitivity of the Arabidopsis triple mutant pyr1/pyl1/pyl4. Taken together, our results suggest that soybean GmPYLs are ABA receptors that function by interacting and inhibiting PP2Cs. PMID:23934343

Abscisic acid (ABA) is a ubiquitous hormone that regulates plant growth, development and responses to environmental stresses. In recent researches, pyrabactin resistance 1-like protein (PYL) and protein phosphatase type 2C (PP2C) were identified as the direct receptor and the second component of ABA signaling pathway, respectively. However, a lot of PYL and PP2C members were found in Arabidopsis and several other plants. Some of them were found not to be involved in ABA signaling. Because of the complex diversity of the genome, few documents have been available on the molecular details of the ABA signal perception system in maize. In the present study, we conducted bioinformatics analysis to find out the candidates (ZmPYL3 and ZmPP2C16) of the PYL and PP2C members most probably involved in ABA signaling in maize, cloned their encoding genes (ZmPYL3 and ZmPP2C16), verified the interaction between these two proteins in response to exogenous ABA induction by yeast two-hybrid assay and bimolecular fluorescence complementation, and investigated the expression patterns of these two genes under the induction of exogenous ABA by real-time fluorescence quantitative PCR. The results indicated that the ZmPYL3 and ZmPP2C16 proteins interacted in vitro and in vivo in response to the induction of exogenous ABA. The downregulated expression of the ZmPYL3 gene and the upregulated expression of the ZmPP2C16 gene are responsive to the induction of exogenous ABA. The ZmPYL3 and ZmPP2C16 proteins are the most probable members of the receptors and the second components of ABA signaling pathway, respectively. PMID:25091169

The development of social behavior is strongly influenced by the serotonin system. Serotonin 2creceptor (5-HT2cR) is particularly interesting in this context considering that pharmacological modulation of 5-HT2cR activity alters social interaction in adult rodents. However, the role of 5-HT2cR in the development of social behavior is unexplored. Here we address this using Htr2c knockout mice, which lack 5-HT2cR. We found that these animals exhibit social behavior deficits as adults but not as juveniles. Moreover, we found that the age of onset of these deficits displays similar timing as the onset of susceptibility to spontaneous death and audiogenic-seizures, consistent with the hypothesis that imbalanced excitation and inhibition (E/I) may contribute to social behavioral deficits. Given that autism spectrum disorder (ASD) features social behavioral deficits and is often co-morbid with epilepsy, and given that 5-HT2cR physically interacts with Pten, we tested whether a second site mutation in the ASD risk gene Pten can modify these phenotypes. The age of spontaneous death is accelerated in mice double mutant for Pten and Htr2c relative to single mutants. We hypothesized that pharmacological antagonism of 5-HT2cR activity in adult animals, which does not cause seizures, might modify social behavioral deficits in Pten haploinsufficient mice. SB 242084, a 5-HT2cR selective antagonist, can reverse the social behavior deficits observed in Pten haploinsufficient mice. Together, these results elucidate a role of 5-HT2cR in the modulation of social behavior and seizure susceptibility in the context of normal development and Pten haploinsufficiency. PMID:26308619

Natriuretic peptide receptor (NPR) A is composed of an extracellular domain (ECD) with a ligand binding site, a single transmembrane region, a kinase homology domain, and a guanylyl cyclase domain. The natural agonists atrial and brain natriuretic peptides (ANP, BNP) bind and activate NPRA, leading to cyclic GMP production, which is responsible for their role in cardiovascular homeostasis. Previous studies suggested that stabilization of a dimeric form of NPRA by agonist is essential for receptor activation. However, ligand specificity and sequential steps of this dimerization process have not been investigated. We used radioligand binding, fluorescence resonance energy transfer homoquenching, and molecular modeling to characterize the interaction of human NPRA-ECD with ANP, BNP, the superagonist (Arg(10),Leu(12),Ser(17),Leu(18))-rANP-(1-28), the minimized analog mini-ANP and the antagonist (Arg(6),beta-cyclohexyl-Ala(8),d-Tic(16),Arg(17),Cys(18))-rANP-(6-18)-amide (A71915). ANP binds to preformed ECD dimers and spontaneous dimerization is the rate-limiting step of the ligand binding process. All the studied peptides, including A71915 antagonist, induce a dose-dependent fluorescence homoquenching, specific to dimerization, with potencies highly correlated with their binding affinities. A71915 induced more quenching than other peptides, suggesting stabilization by the antagonist of ECD dimer in a distinct inactive conformation. In summary, these results indicate that the ligand-induced dimerization process of NPRA is different from that for cytokine receptor model. Agonists or antagonists bind to preformed dimeric ECD, leading to dimer stabilization in an active or inactive conformation, respectively. Furthermore, the highly sensitive fluorescence assay designed to assess dimerization could serve as a powerful tool for further detailing the kinetic steps involved in natriuretic peptide receptor binding and activation. PMID:17965196

Ractopamine (RAC) is fed to an estimated 80% of all beef, swine, and turkey raised in the United States. It promotes muscle mass development, limits fat deposition, and reduces feed consumption. However, it has several undesirable behavioral side effects in livestock, especially pigs, including restlessness, agitation, excessive oral-facial movements, and aggressive behavior. Numerous in vitro and in vivo studies suggest RAC’s physiological actions begin with its stimulation of β1- and β2-adrenergic receptor–mediated signaling in skeletal muscle and adipose tissue; however, the molecular pharmacology of RAC’s psychoactive effects is poorly understood. Using human cystic fibrosis transmembrane conductance regulator (hCFTR) chloride channels as a sensor for intracellular cAMP, we found that RAC and p-tyramine (TYR) produced concentration-dependent increases in chloride conductance in oocytes coexpressing hCFTR and mouse trace amine–associated receptor 1 (mTAAR1), which was completely reversed by the trace amine–associated receptor 1 (TAAR1)–selective antagonist EPPTB [N-(3-ethoxyphenyl)-4-pyrrolidin-1-yl-3-trifluoromethylbenzamide]. Oocytes coexpressing hCFTR and the human β2-adrenergic receptor showed no response to RAC or TYR. These studies demonstrate that, contrary to expectations, RAC is not an agonist of the human β2-adrenergic receptor but rather a full agonist for mTAAR1. Since TAAR1-mediated signaling can influence cardiovascular tone and behavior in several animal models, our finding that RAC is a full mTAAR1 agonist supports the idea that this novel mechanism of action influences the physiology and behavior of pigs and other species. These findings should stimulate future studies to characterize the pharmacological, physiological, and behavioral actions of RAC in humans and other species exposed to this drug. PMID:24799633

Previously we correlated the efficacy for G protein activation with that for arrestin recruitment for a number of agonists at the μ-opioid receptor (MOPr) stably expressed in HEK293 cells. We suggested that the endomorphins (endomorphin-1 and -2) might be biased toward arrestin recruitment. In the present study, we investigated this phenomenon in more detail for endomorphin-2, using endogenous MOPr in rat brain as well as MOPr stably expressed in HEK293 cells. For MOPr in neurons in brainstem locus ceruleus slices, the peptide agonists [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) and endomorphin-2 activated inwardly rectifying K(+) current in a concentration-dependent manner. Analysis of these responses with the operational model of pharmacological agonism confirmed that endomorphin-2 had a much lower operational efficacy for G protein-mediated responses than did DAMGO at native MOPr in mature neurons. However, endomorphin-2 induced faster desensitization of the K(+) current than did DAMGO. In addition, in HEK293 cells stably expressing MOPr, the ability of endomorphin-2 to induce phosphorylation of Ser375 in the COOH terminus of the receptor, to induce association of arrestin with the receptor, and to induce cell surface loss of receptors was much more efficient than would be predicted from its efficacy for G protein-mediated signaling. Together, these results indicate that endomorphin-2 is an arrestin-biased agonist at MOPr and the reason for this is likely to be the ability of endomorphin-2 to induce greater phosphorylation of MOPr than would be expected from its ability to activate MOPr and to induce activation of G proteins. PMID:22553358

Previously we correlated the efficacy for G protein activation with that for arrestin recruitment for a number of agonists at the μ-opioid receptor (MOPr) stably expressed in HEK293 cells. We suggested that the endomorphins (endomorphin-1 and -2) might be biased toward arrestin recruitment. In the present study, we investigated this phenomenon in more detail for endomorphin-2, using endogenous MOPr in rat brain as well as MOPr stably expressed in HEK293 cells. For MOPr in neurons in brainstem locus ceruleus slices, the peptide agonists [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) and endomorphin-2 activated inwardly rectifying K+ current in a concentration-dependent manner. Analysis of these responses with the operational model of pharmacological agonism confirmed that endomorphin-2 had a much lower operational efficacy for G protein-mediated responses than did DAMGO at native MOPr in mature neurons. However, endomorphin-2 induced faster desensitization of the K+ current than did DAMGO. In addition, in HEK293 cells stably expressing MOPr, the ability of endomorphin-2 to induce phosphorylation of Ser375 in the COOH terminus of the receptor, to induce association of arrestin with the receptor, and to induce cell surface loss of receptors was much more efficient than would be predicted from its efficacy for G protein-mediated signaling. Together, these results indicate that endomorphin-2 is an arrestin-biased agonist at MOPr and the reason for this is likely to be the ability of endomorphin-2 to induce greater phosphorylation of MOPr than would be expected from its ability to activate MOPr and to induce activation of G proteins. PMID:22553358

The effect of local administration of the dopamine 2 (DA2)-receptoragonist quinpirole and of the DA1-receptoragonist fenoldopam was studied in the in situ, constant flow autoperfused, superior mesenteric vascular bed of the rat. Local infusion of quinpirole (30 micrograms kg-1 min-1 for 5 min) had no effect on baseline perfusion pressure; it reduced the pressor responses to electrical stimulation (4 Hz, 1 ms, supramaximal voltage) of the periarterial sympathetic nerves to 45.6 +/- 2.1% of its original value but did not modify similar pressor responses produced by locally administered noradrenaline. The inhibitory effect of quinpirole was antagonized by the selective DA2-receptor antagonist domperidone (10 micrograms kg-1) but not by the selective DA1-receptor antagonist SCH 23390 (50 micrograms kg-1). Local infusion of fenoldopam (30 micrograms kg-1 min-1 for 5 min) reduced baseline perfusion pressure to 89.9 +/- 1.9%, increased the pressor response to electrical stimulation (4 Hz, 1 ms, supramaximal voltage) of the periarterial nerves to 134.7 +/- 14.0%, but reduced the pressor response to locally administered noradrenaline to 37.2 +/- 8.2%. Similar pressor responses induced by the selective alpha 1-adrenoceptor agonist phenylephrine were also reduced by fenoldopam (to 38.4 +/- 6.4%), but responses to locally administered angiotensin II were not modified. Pretreatment with SCH 23390 (50 micrograms kg-1) antagonized the effect of fenoldopam on baseline perfusion pressure, but had no influence on the effect of fenoldopam on responses to electrical stimulation or to noradrenaline. Pretreatment with the selective alpha 2-adrenoceptor antagonist rauwolscine (100 micrograms kg-1) had no effect on the reduction in baseline perfusion pressure induced by fenoldopam nor on its inhibitory effect on the response to noradrenaline, but it antagonized the stimulatory effect of fenoldopam on the response to electrical stimulation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2886174

Central neuromedin U 2 receptor (NMU2R) plays important roles in the regulation of food intake and body weight. Identification of NMU2R agonists may lead to the development of pharmaceutical agents to treat obesity. Based on the structure of rutin, a typical flavonoid and one of the NMU2R agonists we previously identified from an in-house made natural product library, 30 flavonoid derivatives have been synthesized and screened on a cell-based reporter gene assay. A number of compounds were found to be selective and highly potent to NMU2R. For example, the EC50 value of compound NRA 4 is very close to that of NMU, the endogenous peptide ligand of NMU2R. Structure-activity relationship analysis revealed that a 3-hydroxyl group in ring C and a 2'-fluoride group in ring B were essential for this class of compounds to be active against NMU2R. PMID:25262941

Peroxisome proliferator-activated receptors (PPARs) are key regulators of lipid and glucose metabolism and have become important therapeutic targets for various diseases. The phytochemical investigation of the chloroform-soluble extract of Chromolaena odorata led to the isolation of a PPAR-gamma agonist, (9 S,13 R)-12-oxo-phytodienoic acid (1), together with 12 other compounds. The structures of chromomoric acid G (2), a new dehydrogenated derivative of 1, and chromolanone (3) were elucidated based on spectroscopic methods. Compound 1 showed a significant effect on PPAR-gamma activation in comparison with rosiglitazone. However, compound 2 was inactive, suggesting that the dehydrogenation of the prostaglandin-like structure in 1 abrogates its PPAR-gamma agonistic activity. PMID:19242902

Calcium-imaging techniques were used to determine if mouse retinal astrocytes in situ respond to agonists of ionotropic (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, AMPA; N-methyl-D-aspartate, NMDA) and metabotropic (S-3,5-dihydroxyphenylglycine, DHPG; trans-1-amino-1,3-cyclopentanedicarboxylic acid, ACPD) glutamate receptors. In most cases we found no evidence that retinal astrocyte intracellular calcium ion concentration ([Ca2+]i) increased in response to these glutamate agonists. The one exception was AMPA that increased [Ca2+]i in some, but not all, mouse retinal astrocytes in situ. However, AMPA did not increase [Ca2+]i in mouse retinal astrocytes in vitro, suggesting that the effect of AMPA in situ may be indirect. PMID:27413752

It has now been demonstrated that the μ, δ1 , δ2 , and κ1 opioid receptor (OR) agonists represent the most promising group of opioids for the creation of drugs enhancing cardiac tolerance to the detrimental effects of ischemia/reperfusion (I/R). Opioids are able to prevent necrosis and apoptosis of cardiomyocytes during I/R and improve cardiac contractility in the reperfusion period. The OR agonists exert an infarct-reducing effect with prophylactic administration and prevent reperfusion-induced cardiomyocyte death when ischemic injury of heart has already occurred; that is, opioids can mimic preconditioning and postconditioning phenomena. Furthermore, opioids are also effective in preventing ischemia-induced arrhythmias. PMID:27197922

Stimulation of toll-like receptor (TLR)9 activates human plasmacytoid dendritic cells and B cells, and induces potent innate immune responses in preclinical tumor models and in patients. CpG oligodeoxynucleotides (ODNs) are TLR9 agonists that show promising results as vaccine adjuvants and in the treatment of cancers, infections, asthma and allergy. PF-3512676 (ProMune) was developed as a TLR9 agonist for the treatment of cancer as monotherapy and as an adjuvant in combination with chemo- and immunotherapy. Phase I and II trials have tested this drug in several hematopoietic and solid tumors. Pfizer has initiated Phase III trials to test PF-3512676 in combination with standard chemotherapy for non-small-cell lung cancer. PMID:17696823

Estrogenic effects are mediated through two estrogen receptor (ER) subtypes, ERα and ERβ. Estrogens are the most commonly prescribed drugs to treat menopausal conditions, but by non-selectively triggering both ERα and ERβ pathways in different tissues they can cause serious adverse effects. The different sizes of the binding pockets and sequences of their activation function domains indicate that ERα and ERβ should have different specificities for ligands and biological responses that can be exploited for designing safer and more selective estrogens. ERα and ERβ regulate different genes by binding to different regulatory elements and recruiting different transcription and chromatin remodeling factors that are expressed in a cell-specific manner. ERα- and ERβ-selective agonists have been identified that demonstrate that the two ERs produce distinct biological effects. ERα and ERβ agonists are promising new approach for treating specific conditions associated with menopause. PMID:20951642

Neuronal nicotinic acetylcholine receptors containing α4, β2, and sometimes other subunits (α4β2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and β2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is β2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4β2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4β2)2 concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but β2 and β4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4β2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets. PMID:25869137

1. BW 723C86 (3 and 10 mg kg-1, s.c. 30 min pretest), a 5-HT2B receptoragonist, increased total interaction, but not locomotion in a rat social interaction test, a profile consistent with anxiolysis. 2. The effect of BW 723C86 in the social interaction test is likely to be 5-HT2B receptor-mediated as it was prevented by pretreatment with the 5-HT2C/2B receptor antagonist, SB 200646A, (1 and 2 mg kg-1, p.o., 1 h pretest) which did not affect basal levels of social interaction at the doses used. 3. An anxiolytic-like action was also observed in the rat Geller-Seifter conflict test, where BW 723C86 (0.5-50 mg kg-1, s.c. 30 min pretest) modestly, but significantly increased punished, but not unpublished responding. 4. In a rat 5 min elevated x-maze test, BW 723C86 (1-10 mg kg-1, s.c.) had no significant effect. 5. The maximal anxiolytic-like effect of BW 723C86 approached that of the benzodiazepine anxiolytic, chloradiazepoxide (5 mg kg-1, s.c. 30 min pretest) in the social interaction test, but was markedly less in the Geller-Siefter test. The effect of BW 723C86 was also clearly less than chlordiazepoxide in the elevated x-maze procedure where it had no significant effect. 6. In conclusion, BW 723C86 exerted an appreciable anxiolytic-like profile in a rat social interaction test, but had a weaker effect in the Geller-Siefter and was ineffective in the elevated x-maze test used. These effects are likely to be 5-HT2B receptor-mediated. PMID:8730737

The biological actions of somatostatin are mediated by a family of five GPCRs, named sst1 to sst5. Somatostatin receptors exhibit equally high-binding affinities to their natural ligand somatostatin-14 and largely overlapping distributions. The overexpression of somatostatin receptors in human tumours is the molecular basis for diagnostic and therapeutic application of the stable somatostatin analogues octreotide, lanreotide and pasireotide. The efficiency of somatostatin receptor signalling is tightly regulated and ultimately limited by the coordinated phosphorylation and dephosphorylation of intracellular carboxyl-terminal serine and threonine residues. Here, we review and discuss recent progress in the generation and application of phosphosite-specific antibodies for human sst2 and sst5 receptors. These phosphosite-specific antibodies are unique tools to monitor the spatial and temporal dynamics of receptors phosphorylation and dephosphorylation. Using a combined approach of phosphosite-specific antibodies and siRNA knock-down screening, relevant kinases and phosphatases were identified. Emerging evidence suggests distinct mechanisms of agonist-selective fine-tuning for individual somatostatin receptors. The recently uncovered differences in phosphorylation and dephosphorylation of these receptors may hence be of physiological significance in mediating responses to acute, persistent or repeated stimuli in a variety of target tissues. PMID:24328848

The trialkyltins tributyltin (TBT) and triphenyltin (TPT) can function as rexinoid-X receptor (RXR) agonists. We recently showed that RXR agonists can alter thyroid hormone (TH) signaling in a mammalian pituitary TH-responsive reporter cell line, GH3.TRE-Luc. The prevalence of TBT and TPT in the environment prompted us to test whether they could also affect TH signaling. Both trialkyltins induced the integrated luciferase reporter alone and potentiated TH activation at low doses. Trimethyltin, which is not an RXR agonist, did not. We turned to a simple, robust, and specific in vivo model system of TH action: metamorphosis of Xenopus laevis, the African clawed frog. Using a precocious metamorphosis assay, we found that 1nM TBT and TPT, but not trimethyltin, greatly potentiated the effect of TH treatment on resorption phenotypes of the tail, which is lost at metamorphosis, and in the head, which undergoes extensive remodeling including gill loss. Consistent with these responses, TH-induced caspase-3 activation in the tail was enhanced by cotreatment with TBT. Induction of a transgenic reporter gene and endogenous collagenase 3 (mmp13) and fibroblast-activating protein-α (fap) genes were not induced by TBT alone, but TH induction was significantly potentiated by TBT. However, induction of other TH receptor target genes such as TRβ and deiodinase 3 by TH were not affected by TBT cotreatment. These data indicate that trialkyltins that can function as RXR agonists can selectively potentiate gene expression and resultant morphological programs directed by TH signaling in vivo. PMID:27167774

The anti-fibrotic, vasodilatory and pro-angiogenic therapeutic properties of recombinant relaxin peptide hormone have been investigated in several diseases, and recent clinical trial data has shown benefit in treating acute heart failure. However, the remodelling capacity of these peptide hormones is difficult to study in chronic settings because of their short half-life and the need for intravenous administration. Here we present the first small-molecule series of human relaxin/insulin-like family peptide receptor 1 agonists. These molecules display similar efficacy as the natural hormone in several functional assays. Mutagenesis studies indicate that the small molecules activate relaxin receptor through an allosteric site. These compounds have excellent physical and in vivo pharmacokinetic properties to support further investigation of relaxin biology and animal efficacy studies of the therapeutic benefits of relaxin/insulin-like family peptide receptor 1 activation. PMID:23764525

Peroxisome proliferator-activated receptor gamma (PPAR{gamma}) regulates metabolic homeostasis and is a molecular target for anti-diabetic drugs. We report here the identification of a steroid receptor ligand, RU-486, as an unexpected PPAR{gamma} agonist, thereby uncovering a novel signaling route for this steroid drug. Similar to rosiglitazone, RU-486 modulates the expression of key PPAR{gamma} target genes and promotes adipocyte differentiation, but with a lower adipogenic activity. Structural and functional studies of receptor-ligand interactions reveal the molecular basis for a unique binding mode for RU-486 in the PPAR{gamma} ligand-binding pocket with distinctive properties and epitopes, providing the molecular mechanisms for the discrimination of RU-486 from thiazolidinediones (TZDs) drugs. Our findings together indicate that steroid compounds may represent an alternative approach for designing non-TZD PPAR{gamma} ligands in the treatment of insulin resistance.

The interactions of dopaminergic agonists and antagonists with /sup 3/H-antagonist labeled D1 dopamine receptors of rat striatum have been characterized. (/sup 3/H)Flupentixol has been found to selectively label D1 dopamine receptors when its binding to D2 dopamine receptors is blocked by the inclusion of D2 selective concentrations of unlabeled spiroperidol or domperidone. Antagonist//sup 3/H-antagonist competition curves are of uniformly steep slope (nH . 1.0) suggesting the presence of a single D1 dopamine receptor. Agonist//sup 3/H-antagonist competition curves are extremely shallow (nH less than or equal to 0.5) for agonists of high relative efficacy, suggesting the presence of heterogeneous populations of agonist-binding states of the D1 dopamine receptor. Computer-modeling techniques were used to estimate affinities and relative site densities for these heterogeneous binding states. This analysis indicates that the ratio of agonist affinities for low and high affinity agonist-binding states is correlated with agonist relative efficacies in activating adenylate cyclase in membrane homogenates. Under the assay conditions employed, the addition of saturating concentrations of guanine nucleotides reduced, but did not abolish, the relative density of high affinity agonist-binding sites. These binding data can, at least in part, be explained by postulating two states of the D1 dopamine receptor, inducible by agonists but not by antagonists and modulated by guanine nucleotides.

Dopaminergic mesolimbic and mesocortical systems are fundamental in hedonia and motivation. Therefore their regulation should be central in understanding depression treatment. This review highlights the dopaminergic activity in relation to depressive behavior and suggests two putative receptors as potential targets for research and development of future antidepressants. In this article we review data that describe the role of serotonin in regulating dopamine release, via 5HT2C and 5HT3 receptors. This action of serotonin appears to be linked to depressive-like behavior and to onset of behavioral effects of antidepressants in an animal model of depression. We suggest that drugs or strategies that decrease 5HT2C and increase 5HT3 receptor-mediated dopamine release in the limbic areas of the brain may provide a fast onset of therapeutic effect. Clinical and basic research data supporting this hypothesis are discussed. PMID:16475958

Retinoic acid (RA) receptor (RAR) agonists are potential toxicants that can cause teratogenesis in vertebrates. To determine the occurrence of RAR agonists in municipal wastewater treatment plants (WWTPs), we examined the RARα agonistic activities of influent and effluent samples from several municipal WWTPs in Osaka, Japan, using a yeast two-hybrid assay. Significant RARα agonistic activity was detected in all the influent samples investigated, suggesting that municipal wastewater consistently contains RAR agonists. Fractionations using high-performance liquid chromatography, directed by the bioassay, found several bioactive peaks from influent samples. The RAR agonists, all-trans RA (atRA), 13-cis RA (13cRA), 4-oxo-atRA, and 4-oxo-13cRA, possibly arising from human urine, were identified by liquid chromatography ion trap time-of-flight mass spectrometry. Quantification of the identified compounds in municipal WWTPs confirmed that they were responsible for the majority of RARα agonistic activity in WWTP influents, and also revealed they were readily removed from wastewater by activated sludge treatment. Simultaneous measurement of the RARα agonistic activity revealed that although total activity typically declined concomitant with the reduction of the four identified compounds, it remained high after the decline of RAs and 4-oxo-RAs in one WWTP, suggesting the occurrence of unidentified RAR agonists during the activated sludge treatment. PMID:22095885

Biased G protein-coupled receptoragonists are orthosteric ligands that possess pathway-selective efficacy, activating or inhibiting only a subset of the signaling repertoire of their cognate receptors. In vitro, d-Trp12,Tyr34-bPTH(7–34) [bPTH(7–34)], a biased agonist for the type 1 PTH receptor, antagonizes receptor-G protein coupling but activates arrestin-dependent signaling. In vivo, both bPTH(7–34) and the conventional agonist hPTH(1–34) stimulate anabolic bone formation. To understand how two PTH receptor ligands with markedly different in vitro efficacy could elicit similar in vivo responses, we analyzed transcriptional profiles from calvarial bone of mice treated for 8 wk with vehicle, bPTH(7–34) or hPTH(1–34). Treatment of wild-type mice with bPTH(7–34) primarily affected pathways that promote expansion of the osteoblast pool, notably cell cycle regulation, cell survival, and migration. These responses were absent in β-arrestin2-null mice, identifying them as downstream targets of β-arrestin2-mediated signaling. In contrast, hPTH(1–34) primarily affected pathways classically associated with enhanced bone formation, including collagen synthesis and matrix mineralization. hPTH(1–34) actions were less dependent on β-arrestin2, as might be expected of a ligand capable of G protein activation. In vitro, bPTH(7–34) slowed the rate of preosteoblast proliferation, enhanced osteoblast survival when exposed to an apoptotic stimulus, and stimulated cell migration in wild-type, but not β-arrestin2-null, calvarial osteoblasts. These results suggest that bPTH(7–34) and hPTH(1–34) affect bone mass in vivo through predominantly separate genomic mechanisms created by largely distinct receptor-signaling networks and demonstrate that functional selectivity can be exploited to change the quality of G protein-coupled receptor efficacy. PMID:23315939

The complete 4775-nt cDNA encoding the human serotonin 5-HT{sub 2C} receptor (5-HT{sub 2C}R), a G-protein-coupled receptor, has been isolated. It contains a 1377-nt coding region flanked by a 728-nt 5{prime}-untranslated region and a 2670-nt 3{prime}-untranslated region. By using the cloned 5-HT{sub 2C}R cDNA probe, the complete human gene for this receptor has been isolated and shown to contain six exons and five introns spanning at least 230 kb of DNA. The coding region of the human 5-HT{sub 2C}R gene is interrupted by three introns, and the positions of the intron/exon junctions are conserved between the human and the rodent genes. In addition, an alternatively spliced 5-HT{sub 2C}R RNA that contains a 95-nt deletion in the region coding for the second intracellular loop and the fourth transmembrane domain of the receptor has been identified. This deletion leads to a frameshift and premature termination so that the short isoform RNA encodes a putative protein of 248 amino acids. The ratio for the short isoform over the 5-HT{sub 2C}R RNA was found to be higher in choroid plexus tumor than in normal brain tissue, suggesting the possibility of differential regulation of the 5-HT{sub 2C}R gene in different neural tissues or during tumorigenesis. Transcription of the human 5-HT{sub 2C}R gene was found to be initiated at multiple sites. No classical TATA-box sequence was found at the appropriate location, and the 5{prime}-flanking sequence contains many potential transcription factor-binding sites. A 7.3-kb 5{prime}-flanking 5-HT{sub 2C}R DNA directed the efficient expression of a luciferase reported gene in SK-N-SH and IMR32 neuroblastoma cells, indicating that is contains a functional promoter. 69 refs., 8 figs., 1 tab.

The receptor channel TRPV1 (Transient Receptor Potential Vanilloid 1) is expressed by primary afferent sensory neurons of the pain pathway, where it functions as a sensor of noxious heat and various chemicals, including eicosanoids, capsaicin, protons and peptide toxins. Comprised of four identical subunits that organize into a non-selective cationic permeable channel, this receptor has a variety of binding sites responsible for detecting their respective agonists. Although its physiological role as a chemosensor has been described in detail, the stoichiometry of TRPV1 activation by its different ligands remains unknown. Here, we combined the use of concatemeric constructs harboring mutated binding sites with patch-clamp recordings in order to determine the stoichiometry for TRPV1 activation through the vanilloid binding site and the outer-pore domain by capsaicin and protons, respectively. We show that, while a single capsaicin-bound subunit was sufficient to achieve a maximal open-channel lifetime, all four proton-binding sites were required. Thus, our results demonstrate a distinct stoichiometry of TRPV1 activation through two of its different agonist-binding domains. PMID:26194846

Atomistic descriptions of the μ-opioid receptor (μOR) noncovalently binding with two of its prototypical morphinan agonists, morphine (MOP) and hydromorphone (HMP), are investigated using molecular dynamics (MD) simulations. Subtle differences between the binding modes and hydration properties of MOP and HMP emerge from the calculations. Alchemical free energy perturbation calculations show qualitative agreement with in vitro experiments performed in this work: indeed, the binding free energy difference between MOP and HMP computed by forward and backward alchemical transformation is 1.2±1.1 and 0.8±0.8 kcal/mol, respectively, to be compared with 0.4±0.3 kcal/mol from experiment. Comparison with an MD simulation of μOR covalently bound with the antagonist β-funaltrexamine hints to agonist-induced conformational changes associated with an early event of the receptor's activation: a shift of the transmembrane helix 6 relative to the transmembrane helix 3 and a consequent loss of the key R165-T279 interhelical hydrogen bond. This finding is consistent with a previous proposal suggesting that the R165-T279 hydrogen bond between these two helices indicates an inactive receptor conformation. PMID:26280453

In light of the evidence that calcium plays a critical role in excitotoxic neuronal death, it has been speculated that the metabotropic glutamate receptor may also contribute to excitotoxic damage through the mobilization of Ca2+ from intracellular stores. In the present study we examined this possibility by studying the neurotoxicity of trans-1-amino-cyclopentyl-1,3-dicarboxylate (trans-ACPD), a selective agonist of the metabotropic glutamate receptor. Exposure of cortical neurons to 100 microM trans-ACPD substantially increased phosphoinositide hydrolysis and intraneuronal free calcium in the presence of CPP and CNQX. Despite the presence of functional metabotropic receptors on cultured neurons, however, exposure of cultures to as high as 1 mM trans-ACPD for 24 h failed to produce any morphological or chemical signs of neuronal damage. Furthermore, trans-ACPD did not potentiate submaximal neurotoxicity produced by other non-N-methyl-D-aspartate (NMDA) agonists, kainate and D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid (AMPA). PMID:1666330

Protease-activated receptor-2 (PAR-2), a receptor activated by trypsin/tryptase, modulates smooth muscle tone and exocrine secretion in the salivary glands and pancreas. Given that PAR-2 is expressed throughout the gastrointestinal tract, we investigated effects of PAR-2 agonists on mucus secretion and gastric mucosal injury in the rat. PAR-2–activating peptides triggered secretion of mucus in the stomach, but not in the duodenum. This mucus secretion was abolished by pretreatment with capsaicin, which stimulates and ablates specific sensory neurons, but it was resistant to cyclo-oxygenase inhibition. In contrast, capsaicin treatment failed to block PAR-2–mediated secretion from the salivary glands. Intravenous calcitonin gene–related peptide (CGRP) and neurokinin A markedly elicited gastric mucus secretion, as did substance P to a lesser extent. Specific antagonists of the CGRP1 and NK2, but not the NK1, receptors inhibited PAR-2–mediated mucus secretion. Pretreatment with the PAR-2 agonist strongly prevented gastric injury caused by HCl-ethanol or indomethacin. Thus, PAR-2 activation triggers the cytoprotective secretion of gastric mucus by stimulating the release of CGRP and tachykinins from sensory neurons. In contrast, the PAR-2–mediated salivary exocrine secretion appears to be independent of capsaicin-sensitive sensory neurons. PMID:11390426

The receptor channel TRPV1 (Transient Receptor Potential Vanilloid 1) is expressed by primary afferent sensory neurons of the pain pathway, where it functions as a sensor of noxious heat and various chemicals, including eicosanoids, capsaicin, protons and peptide toxins. Comprised of four identical subunits that organize into a non-selective cationic permeable channel, this receptor has a variety of binding sites responsible for detecting their respective agonists. Although its physiological role as a chemosensor has been described in detail, the stoichiometry of TRPV1 activation by its different ligands remains unknown. Here, we combined the use of concatemeric constructs harboring mutated binding sites with patch-clamp recordings in order to determine the stoichiometry for TRPV1 activation through the vanilloid binding site and the outer-pore domain by capsaicin and protons, respectively. We show that, while a single capsaicin-bound subunit was sufficient to achieve a maximal open-channel lifetime, all four proton-binding sites were required. Thus, our results demonstrate a distinct stoichiometry of TRPV1 activation through two of its different agonist-binding domains. PMID:26194846

Structure-activity studies of the insulin superfamily member, relaxin-3, have shown that its G protein-coupled receptor (RXFP3) binding site is contained within its central B-chain α-helix and this helical structure is essential for receptor activation. We sought to develop a single B-chain mimetic that retained agonist activity. This was achieved by use of solid phase peptide synthesis together with on-resin ruthenium-catalyzed ring closure metathesis of a pair of judiciously placed i,i+4 α-methyl, α-alkenyl amino acids. The resulting hydrocarbon stapled peptide was shown by solution NMR spectroscopy to mimic the native helical conformation of relaxin-3 and to possess potent RXFP3 receptor binding and activation. Alternative stapling procedures were unsuccessful, highlighting the critical need to carefully consider both the peptide sequence and stapling methodology for optimal outcomes. Our result is the first successful minimization of an insulin-like peptide to a single-chain α-helical peptide agonist which will facilitate study of the function of relaxin-3. PMID:27464307

Antipsychotic drugs provide limited efficacy for cognitive impairment in schizophrenia. Recent studies have found that the neurotensin NTS1 receptoragonist and putative atypical antipsychotic drug PD149163 reverses deficits in sensory-gating and novel object recognition, suggesting that this compound may have the potential to improve cognitive functioning in schizophrenia. The present study sought to extend these investigations by evaluating the effects of PD149163 on sustained attention using a visual signal detection operant task in rats. PD149163, the atypical antipsychotic drug clozapine, and the dopamine D2/3 receptor antagonist raclopride all significantly decreased percent “hit” accuracy, while none of these compounds altered “correct rejections” (compared to vehicle control). Clozapine and raclopride significantly increased response latency, while high doses of PD149163 and raclopride significantly increased trial omissions. Nicotine, which was tested as a positive control, significantly improved overall performance in this task and did not affect response latency or trial omissions. The present findings suggest that neurotensin NTS1 receptoragonists, like antipsychotic drugs, may inhibit sustained attention in this task despite having different pharmacological mechanisms of action. PMID:24076181

The wide ubiquity of GLP-1 receptors in the body has stimulated the search for different extrapancreatic actions of GLP-1 and its receptoragonists. Thus, severe cardioprotective effects directed on myocardial ischaemia and dysfunction as well as diverse antiaterogenic actions have been reported. Also, native and GLP-1 receptoragonists have demonstrated significant beneficial effects on liver steatosis and fibrosis and on neuronal protection in experimental models of Alzheimer, and Parkinson's disease as well as on cerebral ischaemia. Recent evidences suggest that these drugs may also be useful for prevention and treatment of diabetic retinopathy, nephropathy and peripheral neuropathy. Good results have also been reported in psoriasis. Despite we still need confirmation that these promising effects can be applied to clinical practice, they offer new interesting perspectives for treatment of type 2 diabetes associated complications and give to GLP-1 receptoragonists an even more integral position in diabetes therapy. PMID:25437463

Three series of substituted anti-1,2,3-triazoles (IND, PPRD, and QND), synthesized by cycloaddition from azide and alkyne building blocks, were designed to enhance selectivity and potency profiles of a lead α7 nicotinic acetylcholine receptor (α7-nAChR) agonist, TTIn-1. Designed compounds were synthesized and screened for affinity by a radioligand binding assay. Their functional characterization as agonists and antagonists was performed by fluorescence resonance energy transfer assay using cell lines expressing transfected cDNAs, α7-nAChRs, α4β2-nAChRs, and 5HT3A receptors, and a fluorescence cell reporter. In the IND series, a tropane ring of TTIn-1, substituted at N1, was replaced by mono- and bicyclic amines to vary length and conformational flexibility of a carbon linker between nitrogen atom and N1 of the triazole. Compounds with a two-carbon atom linker optimized binding with Kd's at the submicromolar level. Further modification at the hydrophobic indole of TTIn-1 was made in PPRD and QND series by fixing the amine center with the highest affinity building blocks in the IND series. Compounds from IND and PPRD series are selective as agonists for the α7-nAChRs over α4β2-nAChRs and 5HT3A receptors. Lead compounds in the three series have EC50's between 28 and 260 nM. Based on the EC50, affinity, and selectivity determined from the binding and cellular responses, two of the leads have been advanced to behavioral studies described in the companion article (DOI: 10.1021/acschemneuro.5b00059). PMID:25932897

1. The effect of a novel, highly potent and selective kappa-opioid receptoragonist, GR89696, has been evaluated in two animal models of cerebral ischaemia: transient bilateral carotid artery occlusion in the Mongolian gerbil and permanent, unilateral middle cerebral artery occlusion in the mouse. 2. In the Mongolian gerbil model, administration of GR89696 (3 to 30 micrograms kg-1, s.c.), immediately before and at 4 h after insult, produced a dose-dependent reduction in the hippocampal CA1 neuronal cell loss resulting from a 7-min bilateral carotid occlusion. Similar effects were obtained with two other kappa-agonists, GR86014 (1 mgkg-1, s.c.) and GR91272 (1 mgkg-1, s.c.). The neuroprotective effect of GR89696 was completely blocked by prior administration of the opioid receptor antagonist, naltrexone, at 10 mgkg-1, s.c. Repeated post-treatment with GR89696 (100 micrograms kg-1, s.c.) or GR44821 (10 mgkg-1, s.c.) was also effective in protecting completely the hippocampal CA1 neurones from ischaemia-induced neurodegeneration. 3. In the permanent, unilateral middle cerebral artery occlusion model in the mouse, repeated administration of GR89696 at 300 micrograms kg-1, s.c. produced a 50% reduction in cerebrocortical infarct volume. In these experiments GR89696 was dosed 5 min, 4, 8, 12, 16, 20 and 24 h after occlusion on the first day and then three times daily for the next three days. GR89696 (300 micrograms kg-1) also produced a significant 35% reduction in infarct volume in this model when the initiation of dosing was delayed for 6 h after the insult. 4. The results indicate that the potent kappa-opioid receptoragonist, GR89696, is neuroprotective in both global and focal cerebral ischaemia models and suggest that, with this class of compound, there may be a considerable time window for pharmacological intervention. PMID:1657267

Bombesin receptor subtype-3 (BRS-3) is an orphan G protein-coupled receptor in the bombesin receptor family that still awaits identification of its natural ligand. BRS-3 deficient mice develop a mild late-onset obesity with metabolic defects, implicating BRS-3 plays a role in feeding and metabolism. We describe here the pharmacological characterization of a synthetic compound, 16a, which serves as a potent agonist for BRS-3. This compound is selective for BRS-3 as it does not activate neuromedin B or gastrin-releasing peptide receptors, two most closely related bombesin receptors, as well as a series of other GPCRs. We assessed the receptor trafficking of BRS-3 and found that compound 16a promoted β-arrestin translocation to the cell membrane. Neither central nor peripheral administration of compound 16a affects locomotor activity in mice. Therefore compound 16a is a potential tool to study the function of the BRS-3 system in vitro and possibly in vivo. PMID:19580790

Acylureas and acyclic imides are found to be excellent isosteres for 2-acylamino-1,3,4-thiadiazole in the azaxanthene-based series of glucocorticoid receptor (GR) agonists. The results reported herein show that primary acylureas maintain high affinity and selectivity for GR while providing improved CYP450 inhibition and pharmacokinetic profile over 2-acylamino-1,3,4-thiadiazoles. General methods for synthesis of a variety of acylureas and acyclic imides from a carboxylic acid were utilized and are described. PMID:24980053

Patients with cluster headaches occasionally fail to respond to conventional preventive treatments. We herein report a case of a patient with a cluster headache in which the symptoms were refractory to conventional preventive treatments except for high-dose glucocorticoids. The headache attacks occurred daily while sleeping, thus the patient suffered from insomnia. Ramelteon, a selective melatonin receptoragonist and a member of a new class of insomnia therapies, completely suppressed the attacks during sleep and provided rapid relief from insomnia. This is the first English case report to describe the efficacy of ramelteon as a preventive treatment for cluster headaches. PMID:27580554

Parkinson's disease (PD) is a neurodegenerative disease affecting more than a million people in the USA alone. While there are effective symptomatic treatments for PD, there is an urgent need for new therapies that slow or halt the progressive death of dopaminergic neurons. Significant progress has been made in understanding the pathophysiology of PD, which has substantially facilitated the discovery efforts to identify novel drugs. The tissue-protective erythropoietin (EPO) receptor, EPOR/CD131, has emerged as one promising target for disease-modifying therapies. Recombinant human EPO (rhEPO), several variants of EPO, EPO-mimetic peptides, cell-based therapies using cells incubated with or expressing EPO, gene therapy vectors encoding EPO, and small molecule EPO mimetic compounds all show potential as therapeutic candidates. Agonists of the EPOR/CD131 receptor demonstrate potent anti-apoptotic, antioxidant, and anti-inflammatory effects and protect neurons, including dopaminergic neurons, from diverse insults in vitro and in vivo. When delivered directly to the striatum, rhEPO protects dopaminergic neurons in animal models of PD. Early-stage clinical trials testing systemic rhEPO have provided encouraging results, while additional controlled studies are required to fully assess the potential of the treatment. Poor CNS availability of proteins and challenges related to invasive delivery limit delivery of EPO protein. Several variants of EPO and small molecule agonists of the EPO receptors are making progress in preclinical studies and may offer solutions to these challenges. While EPO was initially discovered as the primary modulator of erythropoiesis, the discovery and characterization of the tissue-protective EPOR/CD131 receptor offer an opportunity to selectively target the neuroprotective receptor as an approach to identify disease-modifying treatments for PD. PMID:25832721

The search for synthetic analogues of somatostatin which exhibit selective affinities for the five receptor subtypes is of considerable basic and therapeutic interest and has generated a large number of potent agonist analogues with a wide spectrum of binding profiles. In the past, conformational restriction of side chain groups and the peptide backbone has yielded the most interesting results. Under the latter category and as part of the present study, we were interested in the potential effects of N-methylation of peptide bond NH groups on binding affinity since this approach had not been systematically examined with these peptides. This was aided by new chemistries for introducing an N-Me group during regular solid-phase peptide synthesis using Boc protection. A number of interesting effects were noted on relative binding affinities of the two series of agonist sequences chosen, DPhe(5)(or Tyr(5))-c[Cys(6)-Phe(7)-DTrp(8)-Lys(9)-Thr(10)-Cys(11)]Thr(12)-NH(2) (SRIF numbering), at the five known human somatostatin receptors transfected into and stably expressed by CHO cells. N-Methylation of residues 7 (Phe), 10 (Thr), 11 (Cys), and 12 (Thr) largely destroyed affinities for all five receptors. N-Methylation of DTrp in the DPhe series gave an analogue with extraordinarily high affinity for the type 5 receptor for which it was also quite selective. N-Methylation of Lys in both series resulted in retention of type 2 affinity despite this residue constituting the "active center" of somatostatin peptides. N-Methylation of either the N-terminal Tyr residue or of Cys(6) in the Tyr series resulted in analogues with extraordinarily high affinity for the type 3 receptor, also with a degree of specificity. N-Methylation of the peptide bond constrains the conformational space of the amino acid and eliminates the possibility of donor hydrogen bond formation from the amide linkage. The beta-bend conformation of the agonists around DTrp-Lys is stabilized by a transannular

The Gαi/o-coupled dopamine D2-like receptor family comprises three subtypes: the D2 receptor (D2R), with short and long isoform variants (D2SR and D2LR), D3 receptor (D3R), and D4 receptor (D4R), with several polymorphic variants. The common overlap of norepinephrine innervation and D2-like receptor expression patterns prompts the question of a possible noncanonical action by norepinephrine. In fact, previous studies have suggested that norepinephrine can functionally interact with D4R. To our knowledge, significant interactions between norepinephrine and D2R or D3R receptors have not been demonstrated. By using radioligand binding and bioluminescent resonance energy transfer (BRET) assays in transfected cells, the present study attempted a careful comparison between dopamine and norepinephrine in their possible activation of all D2-like receptors, including the two D2R isoforms and the most common D4R polymorphic variants. Functional BRET assays included activation of G proteins with all Gαi/o subunits, adenylyl cyclase inhibition, and β arrestin recruitment. Norepinephrine acted as a potent agonist for all D2-like receptor subtypes, with the general rank order of potency of D3R > D4R ≥ D2SR ≥ D2L. However, for both dopamine and norepinephrine, differences depended on the Gαi/o protein subunit involved. The most striking differences were observed with Gαi2, where the rank order of potencies for both dopamine and norepinephrine were D4R > D2SR = D2LR > D3R. Furthermore the results do not support the existence of differences in the ability of dopamine and norepinephrine to activate different human D4R variants. The potency of norepinephrine for adrenergic α2A receptor was only about 20-fold higher compared with D3R and D4R across the three functional assays. PMID:26843180

The Gαi/o-coupled dopamine D2-like receptor family comprises three subtypes: the D2 receptor (D2R), with short and long isoform variants (D2SR and D2LR), D3 receptor (D3R), and D4 receptor (D4R), with several polymorphic variants. The common overlap of norepinephrine innervation and D2-like receptor expression patterns prompts the question of a possible noncanonical action by norepinephrine. In fact, previous studies have suggested that norepinephrine can functionally interact with D4R. To our knowledge, significant interactions between norepinephrine and D2R or D3R receptors have not been demonstrated. By using radioligand binding and bioluminescent resonance energy transfer (BRET) assays in transfected cells, the present study attempted a careful comparison between dopamine and norepinephrine in their possible activation of all D2-like receptors, including the two D2R isoforms and the most common D4R polymorphic variants. Functional BRET assays included activation of G proteins with all Gαi/o subunits, adenylyl cyclase inhibition, and β arrestin recruitment. Norepinephrine acted as a potent agonist for all D2-like receptor subtypes, with the general rank order of potency of D3R > D4R ≥ D2SR ≥ D2L. However, for both dopamine and norepinephrine, differences depended on the Gαi/o protein subunit involved. The most striking differences were observed with Gαi2, where the rank order of potencies for both dopamine and norepinephrine were D4R > D2SR = D2LR >> D3R. Furthermore the results do not support the existence of differences in the ability of dopamine and norepinephrine to activate different human D4R variants. The potency of norepinephrine for adrenergic α2A receptor was only about 20-fold higher compared with D3R and D4R across the three functional assays. PMID:26843180

Rationale Chronic treatment with dopamine (DA) receptoragonists and antagonists can differentially affect measures of DA D2/D3 receptor number and function, but the effects of chronic treatment with a partial D2/D3 receptoragonist are not clear. Objective We used a within-subjects design in male cynomolgus monkeys to determine the effects of repeated (17-day) treatment with the D2/D3 receptor partial agonist aripiprazole (ARI; 0.03 mg/kg and 0.1 mg/kg i.m.) on food-reinforced behavior (n=5) and on D2/D3 receptor availability as measured with positron emission tomography (PET; n=9). Methods Five monkeys responded under a fixed-ratio 50 schedule of food reinforcement and D2/D3 receptor availability was measured before and four days after ARI treatment using PET and the D2/D3 receptor-selective radioligand [18F]fluoroclebopride (FCP). Four additional monkeys were studied using [11C]raclopride and treated sequentially with each dose of ARI for 17 days. Results ARI decreased food-maintained responding with minimal evidence of tolerance. Repeated ARI administration increased FCP and raclopride distribution volume ratios (DVRs) in the caudate nucleus and putamen in most monkeys, but decreases were observed in monkeys with the highest baseline DVRs. Conclusions The results indicate that repeated treatment with a low efficacy DA receptor partial agonist produces effects on brain D2/D3 receptor availability that are qualitatively different from those of both high-efficacy receptoragonists and antagonists, and suggest that the observed individual differences in response to ARI treatment may reflect its partial agonist activity. PMID:24077804

Rationale: Serotonin 5-HT2A and 5-HT2Creceptors are thought to be the primary pharmacological mechanisms for serotonin-mediated hallucinogenic drugs, but recently there has been interest in metabotropic glutamate (mGluR2) receptors as contributors to the mechanism of hallucinogens. Objective: The present study assesses the role of these 5-HT and glutamate receptors as molecular targets for two tryptamine hallucinogens, N,N-dimethyltryptamine (DMT) and N,N-diisopropyltryptamine (DiPT). Methods: Drug discrimination, head twitch and radioligand binding assays were used. A 5-HT2AR inverse agonist (MDL100907), 5-HT2CR antagonist (SB242084) and mGluR2/3 agonist (LY379268) were tested for their ability to attenuate the discriminative stimulus effects of DMT and DiPT; an mGluR2/3 antagonist (LY341495) was tested for potentiation. MDL100907 was used to attenuate head twitches induced by DMT and DiPT. Radioligand binding studies and inosital-1-phosphate (IP-1) accumulation were performed at the 5-HT2CR for DiPT. Results: MDL100907 fully blocked the discriminative stimulus effects of DMT, but only partially blocked DiPT. SB242084 partially attenuated the discriminative stimulus effects of DiPT, but produced minimal attenuation of DMT’s effects. LY379268 produced potent, but only partial blockade of the discriminative stimulus effects of DMT. LY341495 facilitated DMT- and DiPT-like effects. Both compounds elicited head twitches (DiPT>DMT) which were blocked by MDL1000907. DiPT was a low potency full agonist at 5-HT2CR in vitro. Conclusions: The 5-HT2AR likely plays a major role in mediating the effects of both compounds. 5-HT2C and mGluR2 receptors likely modulate the discriminative stimulus effects of both compounds to some degree. PMID:24985890

A series of 31 chalcone- and flavonoid-based derivatives were synthesized in good overall yields and screened for their inverse agonist activity on the US28 receptor of human cytomegalovirus (HCMV). With one exception (e.g., 2-(5-bromo-2-methoxyphenyl)-3-hydroxy-4H-chromen-4-one), halogen-substituted flavonoids were typically more potent inverse agonists than their related hydro derivatives. While toxicity could be used to partially explain the inverse agonist activity of some members of the series, 5-(benzyloxy)-2-(5-bromo-2-methoxyphenyl)-4H-chromen-4-one (11b) acted on the US28 receptor as a nontoxic, inverse agonist. The full inverse agonism (efficacy, -89%) and potency (EC50 = 3.5 μM) observed with flavonoid 11b is especially important as it provides both a new tool to study US28 signaling and a potential platform for the future development of HCMV-targeting drugs. PMID:23768434

Human cytochrome P450 (CYP) 2C enzymes metabolize ∼30% of clinically prescribed drugs and various environmental chemicals. CYP2C8, an important member of this subfamily, metabolizes the anticancer drug paclitaxel, certain antidiabetic drugs, and endogenous substrates, including arachidonic acid, to physiologically active epoxyeicosatrienoic acids. Previous studies from our laboratory showed that microRNA 107 (miR107) and microRNA 103 downregulate CYP2C8 post-transcriptionally. miR107 is located in intron 5 of the pantothenate kinase 1 (PANK1) gene. p53 has been reported to coregulate the induction of PANK1 and miR107. Here, we examine the possible downregulation of CYP2C8 by drugs capable of inducing miR107. Hypolipidemic drugs, such as bezafibrate, known activators of the peroxisome proliferator-activated receptor α (PPARα), induce both the PANK1 gene and miR107 (∼2.5-fold) in primary human hepatocytes. Surprisingly, CYP2C8 mRNA and protein levels were induced by bezafibrate. CYP2C8 promoter activity was increased by ectopic expression of PPARα in HepG2 cells, with a further increase after bezafibrate (∼18-fold), 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio acetic acid (∼10-fold) treatment, or the antidiabetic drug rosiglitazone, all known PPAR activators. Promoter sequence analyses, deletion studies, mutagenesis studies, and electrophoretic mobility shift assays identified a PPARα response element located at position -2109 base pair relative to the translation start site of CYP2C8. Chromatin immunopreciptation assay analysis confirmed recruitment of PPARα to this PPARα response element after bezafibrate treatment of human hepatocytes. Thus, we show for the first time that CYP2C8 is transcriptionally regulated by PPARα, suggesting the potential for drug-drug interactions due to upregulation of CYP2C8 by PPAR activators. PMID:26467040

Human cytochrome P450 (CYP) 2C enzymes metabolize ∼30% of clinically prescribed drugs and various environmental chemicals. CYP2C8, an important member of this subfamily, metabolizes the anticancer drug paclitaxel, certain antidiabetic drugs, and endogenous substrates, including arachidonic acid, to physiologically active epoxyeicosatrienoic acids. Previous studies from our laboratory showed that microRNA 107 (miR107) and microRNA 103 downregulate CYP2C8 post-transcriptionally. miR107 is located in intron 5 of the pantothenate kinase 1 (PANK1) gene. p53 has been reported to coregulate the induction of PANK1 and miR107. Here, we examine the possible downregulation of CYP2C8 by drugs capable of inducing miR107. Hypolipidemic drugs, such as bezafibrate, known activators of the peroxisome proliferator-activated receptor α (PPARα), induce both the PANK1 gene and miR107 (∼2.5-fold) in primary human hepatocytes. Surprisingly, CYP2C8 mRNA and protein levels were induced by bezafibrate. CYP2C8 promoter activity was increased by ectopic expression of PPARα in HepG2 cells, with a further increase after bezafibrate (∼18-fold), 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio acetic acid (∼10-fold) treatment, or the antidiabetic drug rosiglitazone, all known PPAR activators. Promoter sequence analyses, deletion studies, mutagenesis studies, and electrophoretic mobility shift assays identified a PPARα response element located at position −2109 base pair relative to the translation start site of CYP2C8. Chromatin immunopreciptation assay analysis confirmed recruitment of PPARα to this PPARα response element after bezafibrate treatment of human hepatocytes. Thus, we show for the first time that CYP2C8 is transcriptionally regulated by PPARα, suggesting the potential for drug-drug interactions due to upregulation of CYP2C8 by PPAR activators. PMID:26467040

Liver X receptor (LXR) plays an important role in reverse cholesterol transport (RCT), and activation of LXR could reduce atherosclerosis. In the present study we used a cell-based screening method to identify new potential LXRβ agonists. A novel benzofuran-2-carboxylate derivative was identified with LXRβ agonist activity: E17110 showed a significant activation effect on LXRβ with an EC50 value of 0.72 μmol/L. E17110 also increased the expression of ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1) in RAW264.7 macrophages. Moreover, E17110 significantly reduced cellular lipid accumulation and promoted cholesterol efflux in RAW264.7 macrophages. Interestingly, we found that the key amino acids in the LXRβ ligand-binding domain had distinct interactions with E17110 as compared to TO901317. These results suggest that E17110 was identified as a novel compound with LXRβ agonist activity in vitro via screening, and could be developed as a potential anti-atherosclerotic lead compound. PMID:27175330

Adenosine A1 receptor (A1AR) agonists have antinociceptive effects in multiple preclinical models of acute and chronic pain. Although numerous A1AR agonists have been developed, clinical applications of these agents have been hampered by their cardiovascular side effects. Herein we report a series of novel A1AR agonists, some of which are structurally related to adenosine 5′-monophosphate (5′-AMP), a naturally occurring nucleotide that itself activates A1AR. These novel compounds potently activate A1AR in several orthogonal in vitro assays and are subtype selective for A1AR over A2AAR, A2BAR, and A3AR. Among them, UNC32A (3a) is orally active and has dose-dependent antinociceptive effects in wild-type mice. The antinociceptive effects of 3a were completely abolished in A1AR knockout mice, revealing a strict dependence on A1AR for activity